Bio
A Resource for Life Science Research Number 109

™
™
Model Isotopic Chemiluminescent Fluorescent Colorimetric
Molecular Imager FX ™ Systems
VersaDoc ™ Imaging Systems
• •
•
•
•
•
•
GS-800 ™ Calibrated Densitometer •
ChemiDoc ™ System
• • •
Gel Doc ™ 2000 System • •
Visit us on the Web at discover.bio-rad.com
Call toll free at 1-800-4BIORAD (1-800-424-6723); outside the US, contact your local sales office.

BioRadiations magazine is published by
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Drive
Hercules, CA 94547 USA
©2002 Bio-Rad Laboratories, Inc.
Copyright reverts to individual authors upon publication.
Reprographic copying for personal use is allowed, provided credit is given to Bio-Rad Laboratories.
Bio-Rad Subsidiaries
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On the cover: Sperm whale myoglobin.
Model courtesy of Accelrys; illustrated by
Audra Geras.
Bio Radiations Volume 109, 2002
Proteomics, the study of protein characteristics and functions, is one of the most exciting and dynamic areas of biological research today, because it offers accessible approaches to a wide spectrum of research fields. Bio-Rad has built on over 45 years of expertise in protein purification and analysis to become a leading supplier of technologies for proteomic research, including the integrated ProteomeWorks ™ system of products. We are committed to providing tools that facilitate proteomic research and its exciting potential applications in drug discovery and other challenging fields.
In this issue’s cover story, we highlight the major subcategories of proteomics, expression proteomics and functional proteomics, and describe the approaches and research tools that are useful in particular types of studies.
Proteomic Solutions: Keys to Enabling Protein Discovery and
Identifying Protein Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
What’s New . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Eve nts Calendar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16
Tips and Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
40
@ the Web . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45
New Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45
Plasmid Purification Using CHT ™ Ceramic Hydroxyapatite Support . . . . . . . .
30
CS Aberin and SG Franklin, Bio-Rad Laboratories, Hercules, CA 94547 USA
Real-Time Immuno-PCR on the iCycler iQ ™ System . . . . . . . . . . . . . . . . . . . . .
32
K Lind and M Kubista, TATAA Biocenter, Göteborg, Sweden
Quantitation of Lymphangiogenesis Using the iCycler iQ ™ Real-Time PCR
Detection System and Scorpions Detection System . . . . . . . . . . . . . . . . . . . . .
34
G Cunnick and WG Jiang, Metastasis Research Group, University Department of Surgery,
University of Wales College of Medicine, Cardiff, UK
Delivery of pCMV-S DNA Using the Helios ® Gene Gun System Is Superior to
Intramuscular Injection in Balb/c Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
A Conn, L Durrant, and I Spendlove, Cancer Research Campaign Academic Unit, Nottingham City
Hospital, Nottingham NG5 1PB, UK
The Versatile Molecular Imager FX ™ Imaging Product Family . . . . . . . . . . . . .
17
Fraction Collection to Microplates With the BioFrac ™ Fraction Collector . . .
20
Bio Radiations Volume 109 3

what’s new
™
Protein Standards Have Never Looked Better
250 kD
150
100
75
50
37
25
20
15
10
Precision Plus
Protein
Unstained
Standards
Precision Plus
Protein
All Blue
Standards
Precision Plus
Protein
Dual Color
Standards
StrepTactin Detection System Overview
Precision Plus Protein unstained standards have the integral affinity Strep -tag amino acid sequence, which binds to StrepTactin, a genetically modified form of streptavidin. This feature allows detection of the standards directly on your blot (Figures 1 and 2).
It is a simple, reliable system, requiring no additional steps. The
StrepTactin-HRP or -AP conjugate is added at the same time as your secondary antibody conjugate. Bio-Rad’s Immun-Star ™ HRP and
Immun-Star AP substrates and reagents allow visualization of signal.
Using this system, you can visualize your Precision Plus Protein unstained standards together with your sample (Figure 1), saving time and giving you accurate results. The standards are compatible with imagers such as the VersaDoc ™ and ChemiDoc ™ systems.
Precision Plus Protein standard with integral
Strep -tag sequence
Add StrepTactin-
AP or -HRP conjugate
Precision Plus Protein standards are available in all blue, dual color, and unstained formulations for all your electrophoresis and blotting needs. Precision Plus Protein standards offer lot-to-lot consistency, unsurpassed band sharpness, and accurate molecular weight determination. The molecular weight of each protein in the unstained standards is confirmed by mass spectrometry.
Comigration and alignment of the prestained (all blue and dual color) and unstained standards is also a key benefit. Each band in the unstained protein standards has been engineered to contain the unique Strep -tag affinity peptide, which allows detection and molecular weight determination directly on western blots.
Precision Plus Protein standards consist of ten highly purified recombinant proteins in the range of 10 to 250 kD, and include three reference bands (25, 50, and 75 kD) for instant orientation.
The dual color standards contain two pink reference bands at 25 and 75 kD and one blue reference band at 50 kD. The all blue standards have three higher-intensity blue reference bands.
1 2 3 4 5 6 7
Fig. 1.
Blot detection. Lane 1, 10 µl of a 1:40 dilution of Precision Plus Protein unstained standards; lanes 3–7, dilutions of transferrin from 80 to 2.5 ng. The blot was incubated with a 1:15,000 dilution of StrepTactin-HRP and a 1:6,000 dilution of anti-transferrin antibody and detected using Immun-Star ™
HRP chemiluminescent substrate. The membrane was exposed to film for 5 sec.
Add substrate
Substrate converted to colored compound or light signal
StrepTactin conjugate binds to Strep -tag sequence
Substrate comes into contact with
StrepTactin-AP or -HRP conjugate
Colored compound or light signal allows visualization of band
Fig. 2.
Overview of the
StrepTactin detection system.
Ordering Information
Catalog #
161-0363
161-0374
161-0373
161-0380
161-0382
170-5040
170-5012
Description
Precision Plus Protein Unstained Standards, 1,000 µl
Precision Plus Protein Dual Color Standards, 500 µl
Precision Plus Protein All Blue Standards, 500 µl
Precision Protein StrepTactin-HRP Conjugate, 0.3 ml
Precision Protein StrepTactin-AP Conjugate, 0.3 ml
Immun-Star HRP Substrate, 500 ml
Immun-Star AP Substrate Pack
Strep -tag and StrepTactin are trademarks of Institut für Bioanalytik GmbH.
Strep -tag technology for western blot detection is covered by US patent
5,506,121, and by UK patent 2,272,698.
4 Bio Radiations Volume 109

™
Simultaneous Removal of Albumin and
Immunoglobulin G (IgG)
The Aurum serum protein mini kit was specifically designed to maximize resolution of proteins of interest in serum, vastly improving and supporting protein discovery and identification.
The isolation of lower abundance proteins from serum or plasma is often complicated by the presence of albumin and IgG, two proteins that can mask the presence of less-abundant comigrating proteins on a 2-D gel.
The Aurum serum protein kit includes Micro Bio-Spin ™ columns filled with a mixture of Affi-Gel ® Blue and Affi-Gel protein A media. This blend of media allows selective binding and simultaneous removal of both albumin and IgG from serum or plasma samples prior to 2-D gel electrophoresis, minimizing handling steps and increasing efficiency. Treated samples are ready for 2-D analysis with no additional purification.
Ordering Information
Catalog #
732-6701
Description
Aurum Serum Protein Mini Kit, includes 10 purification columns, 10 clear 12 x 75 mm polystyrene tubes, 30 sample collection tubes, 10 column tips, 50 ml binding buffer, protocol overview, instructions
For more information, request bulletin 2823 on the reader response card.
™
ReadyStrip IPG strips are now available in a 24 cm length. This new length increases overall resolution in the IEF dimension and, when combined with large format SDS-PAGE in the PROTEAN Plus ™ Dodeca ™ cell, delivers more data from each 2-D gel. The new 24 cm strips, like the other ReadyStrip lengths, are available in broad, narrow, and micro-range pH gradients. Use the broad-range gradients to view your entire sample on one gel, and the narrow and micro-range gradients for more detailed analyses.
7 cm
11 cm
18 cm
17 cm
24 cm
Ordering Information
ReadyStrip IPG Strips, 12 per Package NEW pH Range 7 cm 11 cm 17 cm 18 cm 24 cm
3–10 163-2000
3–10 NL* 163-2002
3–6
4–7
163-2003
163-2001
163-2014
163-2016
163-2017
163-2015
163-2007
163-2009
163-2010
163-2008
163-2032
163-2033
163-2035
163-2034
163-2042
163-2043
163-2045
163-2044
5–8
7–10
3.9–5.1
4.7–5.9
5.5–6.7
6.3–8.3
163-2004
163-2005
163-2028
163-2029
163-2030
163-2031
163-2018
163-2019
163-2024
163-2025
163-2026
163-2027
163-2011
163-2012
163-2020
163-2021
163-2022
163-2023
163-2036
163-2037
163-2038
163-2039
163-2040
163-2041
163-2046
163-2047
163-2048
163-2049
163-2050
163-2051
Catalog # Description
PROTEAN IEF Cell Accessories for 24 cm Strips
165-4042
165-4043
165-4050
24 cm Focusing Tray With Lid, 1
Disposable Rehydration/Equilibration Tray With Lid, 24 cm, 25
Cup Loading Tray Set**, includes 1 tray base, 1 pair electrodes,
1 pack each large and small replacement cups
* Nonlinear
**Recommended for pH range 7–10 and 6.3–8.3 ReadyStrip IPG strips.
Bio Radiations Volume 109 5

what’s new
™
Create, Image, and Analyze DNA and Colony Microarrays
VersArray ChipReader ™ System
The VersArray ChipReader system takes microarray scanning to the next level with superb sensitivity and real-time acquisition analysis, improving productivity and image integrity. Image acquisition is optimized at the low end of the sensitivity range with dual independently controllable lasers and detectors, superior optics, and advanced image processing. This instrument can read two fluorophores at user-selected scanning speeds and resolutions for a variety of slide sizes and substrates. The use of lower laser power and independent control of each channel means reduced noise and higher sensitivity; it also means longer dye lifetime and reduced photobleaching, which allow more scans. Other features include:
• Capability to adjust and optimize scanning parameters in real time
• High-speed laser gating for fast performance without interchannel cross-talk, and simultaneous scanning for greater speed
• Modular slide holder to accommodate most slide configurations, with or without cover glass, wet or dry
Specifications
Chip size
Scan area
Read time
Read sequence
Scan resolution
Dynamic range
Laser information
Excitation wavelengths
Dimensions
25 x 75 mm (1 x 3") or other similar sizes and depths
Up to 22 x 65 mm (0.87 x 2.56"); user-defined area
Approx. 8 min per slide at 10 µm resolution
Simultaneous or independent
3 models: 10–80 µm in 10 µm increments, 5–40 µm in 5 µm increments, 3–24 µm in 3 µm increments
16 bits per pixel, linear detection over the entire range
Two solid-state, 10 mW; independently controlled from 0 to 100%
532 nm and 635 nm (standard)
28 x 23 x 30 cm (11 x 9 x 12"; D x W x H)
VersArray ChipWriter ™ Compact System
The VersArray ChipWriter Compact system is a high-quality DNA array spotting system offering outstanding performance comparable to most larger systems currently available. This system is attractive if you require a smaller arrayer for custom runs, to fit in a limited space, or to fit your budget without compromising quality.
• 24 slide (1 x 3") platen capacity with variety of substrates
• 5–66,000 spots per slide
• Easy-to-use graphical user interface and export of spot information to microarray analysis software
• Available as microarray slide spotting system, membrane gridding system, or both
• Printhead resolution of 1.22 µ m (x, y) and 0.61 µ m (z)
• Monitored environmental control cabinet for accurate results
Specifications
Compatible slide sizes
Slide capacity
25 x 25 mm, 25 x 75 mm, 50 x 75 mm, 75 x 75 mm
Up to 24 slides including 4 blot slides
Maximum number of spots 66,000 with 4 pins (25 x 75 mm slide)
Spot information Typical 90–105 µm on 140–150 µm centers
(user-defined)
Pin information
Printhead information
Type of source plates
Computer requirements
Weight
Dimensions
Up to 48 TeleChem SMP3 Stealth quill pins or solid pins
1.22 µm (x, y axis), 0.61 µm (z axis)
96-, 384-, or 1,536-well microplates
Pentium III, 500 MHz, 128 MB RAM, 6 GB HD,
Windows 2000 operating system, one free serial port
180 lb (83 kg) including environmental chamber
Approx. 87 x 60 x 67 cm (35 x 24 x 27";
D x W x H) including environmental chamber
6 Bio Radiations Volume 109

VersArray ChipWriter ™ Pro System
Accuracy. Repeatability. Reliability. These are key criteria when choosing your microarray printer. The robotic components of this chip writer provide superior accuracy, meaning less time doing spot finding and gridding during image analysis. Repeatability is achieved by using only the highest quality pins for minimal spotto-spot variation so you can have confidence in your results. The
VersArray ChipWriter Pro system is unmatched in reliability — it’s a true walk-away system. Reliability is not just a convenience; it can save thousands of dollars’ worth of repairs and lost materials.
• High-density printing to >81,000 spots per slide
• Flexibility to select spotting pattern and speed, type and number of source plates, and membranes or slides
• Intuitive graphical user interface and open source code software
• Optional liquid transfer and colony picking modules
• Environmental control cabinet for accurate, reproducible prints
Specifications
Capacity
Substrates
Chip size
Spot information
Pin information
Plate information
Printhead information
Dimensions
Up to 126 slides
Glass, matrix, ceramic, and metals
25 x 75 mm (1 x 3"), 26 x 76 mm, and others
Typical 90–105 µm on 140–150 µm centers (user-defined)
Up to 48 TeleChem SMP3 Stealth quill pins or solid pins
Up to 32 in stacker (96- or 384-well microplates)
1.22 µm (x, y axis), 0.24 µm (z axis) resolution,
3 µm repeatability
102 x 130 x 130 cm (41 x 52 x 52"; D x W x H)
For complete specifications and ordering information, visit us on the Web at www.bio-rad.com/versarray
Pentium is a trademark of Intel Corp. Windows is a trademark of Microsoft Corp.
VersArray ™ Colony Arrayer System
The VersArray colony arrayer is a revolutionary robotic platform specially suited for generating and replicating high-density colony arrays for synthetic genetic array (SGA) analysis, a unique new genomic and proteomic application. In addition, the system can be easily transformed to provide liquid handling capabilities, colony picking, or high-throughput gridding. Using a sophisticated image acquisition system, it will pick and inoculate over 9,000 colonies unattended, freeing you for other tasks. Or, from existing 96- and
384-well microplate arrays, it will rearray over 18,000 samples per hour. Like the VersArray ChipWriter Pro system, the VersArray colony picker and arrayer can function as a liquid handling robot that will transfer and rearray volumes ranging from 0.2 to 250 µ l.
• Replicates or rearrays from microplate to microplate or membrane
• Optional four-level elevator for improved capacity
• Easy transition from application to application
• Easy-to-use graphical user interface
• Environmental cabinet that ensures conditions for consistent, accurate results
Specifications
Robotic performance
Arraying throughput
Pin cleaning
Replicator heads types
Colony picking head
Gridding tools
Gripper assembly
Stacker module
Dimensions
Encoder resolution 1.22 µm (x, y axis), 0.24 µm (z axis),
3 µm repeatability
>23,000 colonies/hr (with 1 min wash cycle-variable)
Wash station (with/without pump), sonicator, air drying unit
96, 384, 768 pins, 0.5 or 1.0 mm
High-speed head with 16 indexable pins
96- or 384-pin heads with 0.5 or 1 mm pins
For plate/lid transfer with up/down motion unit
32 shallow plate capacity arranged in 8-plate stacks
102 x 127 x 143 cm (40.2 x 50.0 x 56.3"; D x W x H)
Bio Radiations Volume 109 7

what’s new
™
™
PDQuest 2-D analysis software now has automated spot matching added to its long list of outstanding features. The automatching advantages extend to integrated use of the ProteomeWorks spot cutter, resulting in improved efficiency of the system. Other workflow enhancements have been made for fluorescent protein stain applications. These new features make this integrated system an even more efficient and productive tool in the ProteomeWorks system.
The new integrated PDQuest and ProteomeWorks spot cutter features include:
• Automatching of analysis image to spot cutter image for automated spot cut targeting
• Flip and rotate features to orient fluorescently stained gels without physical movement of the gel fluorescent stains because the spots are not visible. To improve workflow using fluorescently stained gels, the ProteomeWorks spot cutter has new image processing tools such as flip and rotate. Once a gel is placed on the cutting stage of the spot cutter and an image is acquired, the image can be flipped or rotated to match the image orientation of the original analysis and allow the automatching feature to operate without physically moving the gel.
For more information on PDQuest and the ProteomeWorks spot cutter, visit us on the Web at discover.bio-rad.com
Ordering Information
Catalog #
165-7009
165-7042
170-8603
Description
ProteomeWorks Spot Cutter
ProteomeWorks Spot Cutter With Fluorescent Enclosure
PDQuest 2-D Analysis Software Automatching Enhances the Spot Cutter Workflow
PDQuest automated spot matching improves the speed and efficiency of the spot matching process for comparison of 2-D gels.
The same principle applies to using the PDQuest matchset to target and cut gels on the ProteomeWorks spot cutter. Using automated spot matching to target the spot cutter, the time needed to make spot selections is greatly decreased, while still retaining the ability to use free gels. The software provides important feedback on the confidence of matching, so you always have the ability to control exactly what is going to be cut. You’ll spend minimal time with the spot cutter during setup, and can take advantage of its automated operation more than ever.
SYPRO is a trademark of Molecular Probes, Inc.
Flip and Rotate for Easy Orientation
Fluorescent stains such as SYPRO Ruby protein gel stain are used increasingly in proteomic laboratories as their advantages are more widely realized. The proper orientation of a gel can be difficult with
The PDQuest automatching function extends to automated targeting of the
ProteomeWorks spot cutter.
8 Bio Radiations Volume 109
The flip and rotate functions allow the spot cutter image to be oriented to the original image without moving the gel on the spot cutter platform.

™
Are you still trying to schedule your PCR runs? The iCycler with dual 48-well module is the answer to the needs of your productive laboratory. This module has two independently controlled 48-well blocks using standard 0.2 ml tubes and plates, effectively converting the iCycler into two powerful thermal cyclers.
All iCycler reaction modules are designed as modular upgrades to the iCycler chassis. The addition of the dual 48-well module to existing 60-, 96-, and 384-well reaction modules makes the iCycler the only thermal cycler you’ll ever need. In addition, your iCycler can be directly upgraded to real-time quantitative PCR capability by modular attachment of the 96-well block and optical module.
Other Advantages of the iCycler
• 1/4 VGA screen and intuitive programming make protocol viewing, editing, and running easy
• Modular design supports interchangeable reaction modules
• Maximum sample flexibility accommodates 0.2 ml tubes, strips, and plates, 0.5 ml tubes, and 384-well PCR plates
• Temperature monitoring and control can be specified as in-sample probe, block, or algorithm
• Innovative vertical thermal gradient format for 96-well reaction module provides maximum assay development capabilities
Ordering Information
Catalog #
170-8722
170-8705
170-8720
170-8724
170-8726
170-8703
170-8707
170-8709
223-9473
Description iCycler System With 2 x 48 x 0.2 ml Reaction Module, includes base thermal cycler, in-sample temperature probe, PCR tubes, power cord, quick-reference card, instructions
2 x 48 x 0.2 ml Reaction Module iCycler Thermal Cycler With 96 x 0.2 ml Reaction Module iCycler Thermal Cycler With 60 x 0.5 ml Reaction Module iCycler Thermal Cycler With 384-Well Reaction Module iCycler Thermal Cycler 96 x 0.2 ml Reaction Module iCycler Thermal Cycler 60 x 0.5 ml Reaction Module iCycler Thermal Cycler 384-Well Reaction Module
0.2 ml Thin-Wall Tubes, natural, 1,000
™
®
Bio-Rad introduces the newest addition to its line of quantitative
PCR reagents: iQ SYBR Green supermix, specifically formulated to achieve optimal results in real-time SYBR Green assays. It contains
SYBR Green I dye, hot-start iTaq ™ DNA polymerase, optimized buffer, and dNTPs. The supermix also contains a dilute concentration of fluorescein for well-factor collection on the iCycler iQ ™ real-time PCR detection system. With iQ SYBR
Green supermix, you minimize preparation time and produce reliable results for your real-time PCR assays.
• Contains all reagent components needed for SYBR Green I PCR assays, including iTaq DNA polymerase
• Demonstrated high performance for both human genomic DNA and plasmid DNA over a wide dynamic range
• Buffers optimized for SYBR Green I stability, ensuring reliable performance and minimal optimization
• Qualified for use with the iCycler iQ detection system
To see data generated using the iQ SYBR Green supermix, visit our web site at www.bio-rad.com/iCycler and select “What’s New”. For more information, request bulletin 2846 on the reader response card.
Amplification of the IL-1
β gene from human genomic DNA. Serial dilutions of human genomic DNA (5-fold from 50 ng to 16 pg) were amplified using gene-specific primers.
Standard curve of the serial dilution: r = 0.997, efficiency = 98.4%.
Ordering Information
Catalog#
170-8880
170-8882
Description iQ SYBR Green Supermix, 100 x 50 µl reactions iQ SYBR Green Supermix, 500 x 50 µl reactions
Practice of the patented polymerase chain reaction (PCR) process requires a license.
SYBR is a trademark of Molecular Probes, Inc. Bio-Rad Laboratories is licensed by
Molecular Probes to sell reagents containing SYBR Green I for use in real-time PCR, for research purposes only.
9 Bio Radiations Volume 109

what’s new
™
A New Standard for Personal Thermal Cyclers
Personal thermal cyclers are designed to give quality performance at an economical price. The MyCycler meets these expectations by combining design and interface with performance and price.
This innovative thermal cycler features a large graphical display and intuitive programming, ensuring that your time is spent on evaluating results, not performing tedious setup procedures.
The MyCycler was designed to maximize benchspace usage without influencing throughput. This is achieved by a 96-well x
0.2 ml sample format, supporting reaction volumes of 15–100 µl.
Specifications
Technical
Range
Accuracy
Uniformity
Heated lid
Heating rate
Cooling rate
Method of heating/cooling
Modes of temp. control and monitoring
4–100°C
±0.5°C
±0.5°C
Adjustable to 110°C
2.5°C/sec
1.5°C/sec
Peltier and Joule
Block, in-sample probe, algorithm
Descriptive
Footprint
Sample capacity
Display
Licensed for PCR
20 x 26 x 42 cm
(7.9 x 10.2 x 16.5"; H x W x D)
96 wells x 0.2 ml
4.7" screen; 1/4 VGA resolution
Yes
Programming
Onboard software
Key programming features
Menu driven, real-time graphical display of thermal protocol; intuitive graphical programming, automatic run validation reporting, customizable auditory signals for pause and end of run
Protocol templates for most common applications, such as two- and three-step PCR, cycle sequencing, touchdown PCR, RT-PCR, nested PCR, and long PCR
Ample storage space for user-defined protocols. Information/help built into the software interface
Maximum number of programs 99
Cycles per protocol
Segments per cycle
9
9
Looking for more than a personal thermal cycler? When multiple sample blocks or alternative sample volumes are required, or if faster cycling is preferred, the iCycler ™ thermal cycler offers a comprehensive array of solutions.
The large graphics display and intuitive programming make it easy to edit and evaluate protocols.
For more information, contact your local Bio-Rad representative or visit us on the Web at discover.bio-rad.com
Ordering Information
Catalog #
170-9703
223-9473
Description
MyCycler Thermal Cycler, includes thermal cycler, power cord, quick guide, instructions
0.2 ml Thin-Wall Tubes, natural, 1,000
Practice of the patented polymerase chain reaction (PCR) process requires a license. The MyCycler thermal cycler includes a licensed thermal cycler and may be used with PCR licenses available from Applied Biosystems.
10 Bio Radiations Volume 109

™
•
•
•
•
•
•
•
•
•
•
•
Taq
The polymerase chain reaction (PCR) process is covered by patents owned by Hoffman-LaRoche. Use of the PCR process requires a license.
Visit us on the Web at discover.bio-rad.com
Call toll free at 1-800-4BIORAD (1-800-424-6723); outside the US, contact your local sales office.

what’s new
™
A Choice of Exponential or Square-Wave Pulses
The Gene Pulser Xcell is a compact, versatile electroporation system that uses exponential or square waves to deliver the pulses optimal for your cell type. Both exponentially decaying and squarewave pulses have been used very effectively for both electroporation and electrofusion. The shape of the electroporation wave can have a significant effect on the transformation efficiency for different cell types. The Gene Pulser Xcell system generates both exponential and square waveforms, enabling you to choose the waveform and protocol that will work best for your cells.
Reliable, Reproducible, and Safe Performance With
PulseTrac ™ Circuitry
The unique PulseTrac delivery system ensures that only the highest quality electroporation pulses are consistently delivered while offering maximum sample protection.
User-Friendly Interface Provides Easy Programming
The graphical interface on the main unit controls all functions, including those of accessory modules. It enables easy, intuitive programming using onscreen prompts, and displays all experimental parameters. Programs include:
• Preset optimized programs for rapid program selection for commonly used microbial and mammalian cell lines
• User method storage
• An optimization program to determine ideal conditions
• Storage and recall of pulse parameters and results
Modular Design Enables Choice of Systems
The system is available as a total system, a microbial system, or a eukaryotic system, allowing you to choose exactly what you need for your laboratory.
ShockPod ™ Chamber Designed for One-Handed Use
The system is completed by the innovative ShockPod cuvette chamber, designed for single-handed operation to provide more rapid sample handling.
Whether you are pioneering new applications or reproducing proven protocols, the Gene Pulser Xcell system provides all the features necessary to meet demanding and ever-evolving research needs.
For more information, request bulletin 2750 on the reader response card.
Ordering Information
Catalog #
165-2660
Description
Gene Pulser Xcell Total System, for eukaryotic and microbial cells, 100/240 V, 50/60 Hz, exponential and square-wave delivery, includes main unit, CE module, PC module, ShockPod chamber, 15 sterile cuvettes (5 each of 0.1, 0.2, and 0.4 cm gap), instructions
165-2661
165-2662
165-2666
165-2667
165-2668
165-2669
Gene Pulser Xcell Eukaryotic System, 100/240 V, 50/60 Hz, exponential and square-wave delivery, includes main unit, CE module, ShockPod chamber, 5 sterile cuvettes (0.4 cm gap), instructions
Gene Pulser Xcell Microbial System, 100/240 V, 50/60 Hz, exponential and square-wave delivery, includes main unit, PC module, ShockPod chamber, 10 sterile cuvettes (5 each of 0.1
and 0.2 cm gap), instructions
Gene Pulser Xcell Main Unit, 100/240 V, 50/60 Hz, instructions
Gene Pulser Xcell CE Module, 25–3,275 µF range controlled by main unit, includes integral leads, 5 sterile cuvettes (0.4 cm gap)
Gene Pulser Xcell PC Module, 50–1,000
Ω range controlled by main unit, includes integral leads, 10 sterile cuvettes (5 each of
0.1 and 0.2 cm gap)
Gene Pulser Xcell ShockPod Chamber, includes integral leads for connection to Gene Pulser Xcell, Gene Pulser II, or MicroPulser electroporator
12 Bio Radiations Volume 109

gene
™
™
•
•
•
•
•
•
Electroporation Biolistics Microinjection Lipofection
Visit us on the Web at discover.bio-rad.com
Call toll free at 1-800-4BIORAD (1-800-424-6723); outside the US, contact your local sales office.

what’s new
™
Bio-Rad has significantly extended its gene transfer product portfolio with the introduction of the XenoWorks microinjection system.
Available as a complete system or as individual instruments, XenoWorks offers a complete solution for any microinjection application.
XenoWorks Micromanipulator
The XenoWorks micromanipulator features a hanging joystick controller with a low-profile base that allows comfortable use over extended periods. The stepper-motor positioning system operates over six different ranges, and microtool movement is exceptionally smooth and responsive.
XenoWorks Digital Microinjector
The XenoWorks digital microinjector provides two pressure-control channels, one for holding suspended cells and one for injecting. The injecting channel is capable of providing high-pressure pulses of up to 5,600 hPa, or gentle aspiration and injection for delicate cell manipulation procedures such as embryonic stem (ES) cell transfer.
XenoWorks Analog Microinjector
The XenoWorks analog microinjector is a versatile, low-cost, micrometer-type microinjector for cell-holding or transfer applications. With coarse and fine control, and the ability to use hydraulic oil or air, this microinjector is suitable for intracytoplasmic sperm injection (ICSI), ES cell transfer, and nuclear transfer.
Ordering Information
Catalog #
165-2801
165-2802
165-2805
165-2806
165-2808
165-2830
165-2831
165-2840
165-2842
165-2843
165-2850
165-2852
165-2860
165-2862
Description
XenoWorks Micromanipulator (Left), 110/240 V, 50/60 Hz, includes motor drive, control module, joystick interface, instructions
XenoWorks Micromanipulator (Right), 110/240 V, 50/60 Hz, includes motor drive, control module, joystick interface, instructions
XenoWorks Digital Microinjector, 110 V, 60 Hz, includes compressor module, user interface, 2 microtool holders,
2 digital microinjector tubing kits, instructions
XenoWorks Digital Microinjector, 240 V, 50 Hz, includes compressor module, user interface, 2 microtool holders,
2 digital microinjector tubing kits, instructions
XenoWorks Analog Microinjector, 1,000 µl, includes micrometer drive, microtool holder, analog tubing kit, instructions
Microscope Adaptor, Olympus IX-50/70
Microscope Adaptor, Olympus IX-51/71
Microscope Adaptor, Nikon TMD
Microscope Adaptor, Nikon Diaphot/Eclipse TE 200/300
Microscope Adaptor, Nikon TE 2000
Microscope Adaptor, Zeiss Axiovert 100/135
Microscope Adaptor, Zeiss Axiovert 200
Microscope Adaptor, Leica DM-IL
Microscope Adaptor, Leica DM-IRB/E/-IRE2
14 Bio Radiations Volume 109

gene
™
Whether you’re delivering DNA into a pronucleus or transferring stem cells to a blastocyst, the XenoWorks microinjection system helps you sail through your work.
You’ll feel the difference the moment you take the controls. The ergonomic design with height-adjustable joystick, micromanipulator position memories, and variable movement radius makes the XenoWorks system faster, easier, and more comfortable to guide than other microinjection instruments.
• Air injection system provides exceptional stability and reproducibility
• Intuitive interface (no hierarchical menus or programming)
• Mechanically stable micromanipulator for piezo-drilling applications
For more information, contact your Bio-Rad representative or set course for www.bio-rad.com/genetransfer/
Visit us on the Web at discover.bio-rad.com
Call toll free at 1-800-4BIORAD (1-800-424-6723); outside the US, contact your local sales office.
Microinjection Lipofection Biolistics Electroporation

Below is a list of conferences that will have Bio-Rad representatives available on-site. For an updated list of conferences and other events, visit our web site at discover.bio-rad.com
and click on “events”. Contact your local Bio-Rad representative to let us know of other events of interest to you.
Conference Exhibit Dates
2002
Österreichische Gesellschaft für Molekularbiologie & Gentechnologie
The Botanical Society of Japan
Drei Länder Tagung Lebensmittelhygiene
ComBio 2002
2003
Elettroforesi Bidimensionale, Corso Teorico e Pratico
Association for Biomolecular Research Facilities
Pittsburgh Conference (PittCon)
National Science Teachers Association, National Meeting
Sept 16–18
Sept 21–23
Sept 24–27
Sept 29–Oct 3 iQ-Check Real-TIme PCR Seminar for Food and Water Analysis
BioMalaysia 2002
Genome Sequencing and Analysis Conf/TIGR
I Colori della Real Time PCR, Seminario Verde
Oct 1
Oct 2–4
Oct 2–5
Oct 3
Int’l Symposium on Preparative and Industrial Chromatography and Allied Techniques (SPICA) Oct 6–9
Cytokines and Interferons 2002 Oct 6–10
I Colori della Real Time PCR, Seminario Blu
European Society of Gene Therapy, 10th Annual Meeting
Japan Biochemical Society
New Jersey Science Teachers Association
Oct 10
Oct 12–16
Oct 14–17
Oct 15–16
Carrefour Europeen des Biotechnologies
R&D in Life Sciences
Congrès de la Société Française d’Electrophorese et d’Analyse Protéomique
West Virginia Science Teachers Association
Maryland Association of Science Teachers
Oct 15–16
Oct 15–18
Oct 16–18
Oct 17–19
Oct 18
I Colori della Real Time PCR, Seminario Rosso
Elettroforesi Bidimensionale, Corso Teorico e Pratico
National Science Teachers Association, Eastern Area
California Science Teachers Association
Metropolitan Association of College and Univ Biologists
4th HUGO Pacific Meeting and 5th Asia-Pacific Conference on Human Genetics
Chips to Hits 2002
BioMedical Asia 2002
ASME Bioprocess Technology Seminar
Japan Bioimaging Society
National Association of Biology Teachers
Oct 23
Oct 23–25
Oct 24–26
Oct 24–27
Oct 26
Oct 27–30
Oct 27–31
Oct 28–30
Oct 28–Nov 1
Oct 30–Nov 1
Oct 30–Nov 2
Society for Neuroscience Annual Meeting
AIChE/American Electrophoresis Society Annual Meeting
Het Instrument Exhibition
La PCR nel Nuovo Millennio, 2002
Williamsburg BioProcessing Foundation, Viral Vectors and Vaccines Conference
National Science Teachers Association, Northwestern Area
Biopharmaceutical Production Week
HUPO (Human Proteome Organization) Annual Meeting
Nov 2–7
Nov 4–6
Nov 4–8
Nov 4–8
Nov 11–14
Nov 14–16
Nov 18–21
Nov 21–24
Second Int’l Conference on Structural Biology and Functional Genomics
Swiss Proteomics Society Congress 2002
Elettroforesi Bidimensionale, Corso Teorico e Pratico
National Science Teachers Association, Southwestern Area
Cell Biology
Dec 2–4
Dec 3–5
Dec 4–6
Dec 5–7
Dec 13–18
Feb 5–7
Mar 1–4
Mar 9–14
Mar 27–30
16 Bio Radiations Volume 109
Location
Salzburg, Austria
Kyoto, Japan
Garmisch, Germany
Sydney, Australia
Roozendaal, The Netherlands
Kuala Lumpur, Malaysia
Boston, MA, USA
Roma, Italy
Heidelberg, Germany
Torino, Italy
Bari, Italy
Antibes Juan-les-Pins, France
Kyoto, Japan
Somerset, NJ, USA
Lille, France
Basel, Switzerland
Lille et Villeneuve d’Ascq, France
Charleston, WV, USA
Urbana, MD, USA
Bologna, Italy
Colleretto Giacosa, Italy
Louisville, KY, USA
San Francisco, CA, USA
Brooklyn, NY, USA
Pattaya, Chonburi, Thailand
Philadelphia, PA, USA
Singapore
San Diego, CA, USA
Nagoya, Japan, USA
Cincinnati, OH, USA
Orlando, FL, USA
Indianapolis, IN, USA
Jaarbeurs Utrecht, The Netherlands
Milano, Italy
New Orleans, LA, USA
Portland, OR, USA
San Diego, CA, USA
Versailles, France
Singapore
Lausanne, Switzerland
Colleretto Giacosa, Italy
Albuquerque, NM, USA
San Francisco, CA, USA
Colleretto Giacosa, Italy
Denver, CO, USA
Orlando, FL, USA
Philadelphia, PA, USA

™
In 1998, Bio-Rad introduced the Molecular
Imager FX imaging system. The first product in the Molecular Imager FX line, this sophisticated, state-of-the-art instrument combined fluorescent, storage phosphor, and indirect chemiluminescent imaging (using Bio-Rad StrS screens) in a single high-performance instrument. The Molecular
Imager FX remains a laboratory workhorse, and since then has evolved into an entire family of quality products tailored for specific applications: the Personal Molecular Imager FX ™ system for storage phosphor imaging, the Molecular Imager
FX Pro ™ system for fluorescent imaging, and the
Molecular Imager FX Pro Plus ™ multiimager system for fluorescent and storage phosphor imaging. The detection modes supported by each of these instruments are summarized in Table 1.
The FX family offers you a choice of the best technologies for your application, whether laser excitation for single or multicolor fluorescence detection, or storage phosphor imaging for exceptional sensitivity and resolution of radioisotopes. An optional external laser port that houses up to three lasers allows you to change research focus when needed. You can even create custom applications.
Fast Scanning With the Scan Resolution and Sensitivity That You Need
The FX systems are also designed for reliability and fast, sensitive performance. Molecular Imager FX systems have a unique, fast-scanning mechanism that is based on patented fiber-optic technology optimized for enhanced low-light collection. The systems can accommodate samples up to 35 x
43 cm and offer multiple-resolution scanning, including higher-resolution scanning to 50 µm.
Like all of Bio-Rad’s imaging systems, the FX systems are designed to provide affordable solutions to fit your exact needs. For gel and blot imaging, you don’t need to pay for higher resolution when your applications don’t require it. Gels, blots, and phosphor screens rarely have features that can’t be resolved at a 100 µm scan on a Molecular Imager
FX system. (Figure 1 demonstrates the subtle difference in scanning a resolution target at both
50 µm and 25 µm with a competitor’s imaging system.) The rationale for having the capability of scanning at higher resolution is to enable scanning of microarrays. However, for microarray applications, a dedicated imager such as a
VersArray ChipReader ™ system (see page 6) can allow much higher throughput than a multiimager, because it can scan up to 3 times faster.
For other applications, scan time and file size quickly become unmanageable when scanning at
Molecular Imager FX
Pro Plus System
Table 1. The Molecular Imager FX family of products.
Detection Modes
Supported
Molecular
Imager FX
System
Personal
Molecular
Imager FX
System
Fluorescence
Chemifluorescence
Radioisotopes
Chemiluminescence
•
•
•
•
•
Molecular
Imager FX Pro
Fluorescent
Imager
Molecular
Imager FX
Pro Plus
MultiImager
System
•
•
•
•
•
Bio-Rad Imaging Systems — All Your Needs From One Source
When your research requires imaging capabilities, Bio-Rad’s comprehensive line of imaging systems provides all the features you need. Bio-Rad offers imaging products that utilize a variety of detection modes for a wide range of sample types and applications:
• The GS-800 ™ calibrated densitometer provides quantitative detection and analysis of transparent and opaque samples: gels, colorimetric and film-based chemiluminescent dot and slot blots, autoradiograms, slides, and photographs.
• Molecular Imager ® systems use laserscanning and storage phosphor technology for fluorescent or radioisotopic detection.
These capabilities are ideal for imaging gels, blots, microplates, and TLC plates.
• The ChemiDoc ™ system captures and analyzes fluorescent, chemiluminescent, and colorimetric gel samples.
• The new VersArray ChipReader ™ system uses laser-scanning technology for rapid imaging and analysis of fluorescent microarrays. (For more information on the
VersArray ChipReader and related microarray products, see pages 6–7.)
• VersaDoc ™ imaging systems utilize high-resolution digital CCD technology for quantitation of fluorescent, chemifluorescent, chemiluminescent, and colorimetric gels, blots, microplates, and autoradiograms.
• The Gel Doc 2000 ™ gel documentation system captures gel images in real time, enabling rapid optimization, annotation, analysis, and printing.
All of Bio-Rad’s image acquisition and analysis systems can be easily integrated into a single imaging center and networked for rapid, efficient functionality. Whatever your application and budget, there’s a Bio-Rad imaging system that’s perfect for both your present and future research needs.
Bio Radiations Volume 109 17

very high resolution. For example, scanning a 35 x
43 cm platen at 10 µm on a competitor’s system would take over 7 hours and generate a file size of 2,900 Mb (Table 2), several times the memory available on even the most powerful desktop computer today.
For sensitivity of detection for multiple fluorophores and radioisotopes, it’s hard to beat the Molecular Imager FX product family. Each FX system has even been designed with fluorescence expandability in mind to meet your future research needs. As technology continues to evolve, the range of available fluorescent dyes, labels, and substrates is rapidly expanding. To help you keep pace with these advances, the Molecular Imager
FX utilizes a fully integrated external laser port for an optional add-on external laser module, guaranteeing access to tomorrow’s applications.
Fig. 1.
Similarity of results at 50 µm and 25 µm scan resolutions on a competitor’s system. Differences are only evident in the lines of the target separated by <250 µm, far more than needed to resolve bands.
Fluorescent Imaging
The Molecular Imager FX systems’ chromatically corrected scan head enables detection of a wide range of fluorescent dyes or labels, including Cy, ethidium bromide, fluorescein, Radiant ® Red, rhodamine, SYBR Green I, SYPRO Ruby, and
Texas Red dyes. (Request bulletin 2421 for fluorophore excitation and emission data.)
The use of multiple lasers provides flexibility and upgradability by allowing optimal excitation of single or multicolor fluorescent samples. A unique dual-wavelength internal laser can be
50 µm scan
25 µm scan
18 mm 5 mm
250 µm
500 µm
250 µm
500 µm supplemented by an optional add-on external laser module housing up to three laser lines. The optional external laser source has rapid, simple, and safe user coupling. It has motorized autoalignment of the fiber-optic connection. The high coupling efficiency ensures optimal detection sensitivity. This field upgrade guarantees that you can change research focus or take advantage of developments in fluorescent dye technology.
Molecular Imager FX fluorescence analysis is exceptionally easy, with all fluorophore detection parameters automatically selected by intuitive, application-oriented software. Software-controlled filter wheels accommodate eight filter slots, allowing detection of combinations of dyes. The scan head is located above the sample to increase collection efficiency and reduce autofluorescence.
Radioisotopic Imaging
Molecular Imager FX systems’ storage phosphor imaging technology offers the ultimate in isotopic imaging: unsurpassed sensitivity, exceptional resolution, and time-saving exposures that are at least 10 times faster than traditional film techniques. With a linear dynamic range far exceeding that of X-ray film, Bio-Rad’s Molecular
Imager FX family provides superior quantitation over a large dynamic range — up to 5 orders of magnitude. A wide selection of research isotopes can be analyzed, including 3 H, 14 C, 32 P, 33 P, and
35 S. It’s also compatible with most commercially available phosphor screens, including those manufactured by Kodak and Fuji.
User-Friendly, Multiplatform Software
All application configurations are driven by easyto-use Quantity One ® software, compatible with
Windows NT, Windows 98, Windows 2000, or
Macintosh OS 9 operating systems. Simply choose the application, and the software automatically selects the optimal settings (laser, filter, etc.).
Users familiar with the software for the FX product family will find that the same intuitive software also runs other Bio-Rad imagers.
After an image has been acquired, quantitate it with powerful Quantity One analysis tools.
The software includes turn-key application templates for most common fluorophores. In addition, there are application-specific programs for fingerprinting, 2-D, and sequencing analysis.
You can also create custom applications. For an example of multicolor fluorescent imaging capabilities, request bulletin 2597.
18 Bio Radiations Volume 109

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Measuring Up to the Competition
The Molecular Imager FX Pro Plus was put through parallel testing with comparable imaging systems from other major manufacturers. Each of the systems was tested for sensitivity with common fluorescent and isotope imaging applications and all of the imaging systems gave excellent results. Figures 2 and 3 show sample results from the Molecular Imager FX Pro and competitor systems. Additional considerations — the faster scan times, lighter weight, and more compact dimensions of the Molecular Imager FX
Pro Plus — make it a superior value for your research dollar (instrument specifications are listed in Table 3). For more specialized requirements, consider the Molecular Imager FX
Pro (fluorescence imaging) or Personal Molecular
Imager FX systems (radioisotope imaging). For more information about the Molecular Imager
FX family, request bulletin 2737.
You may be considering the purchase of multiple Bio-Rad imaging instruments — to have application-dedicated imagers, for example, or to establish an integrated laboratory imaging center.
If so, you can take advantage of personalized, bundled system packages to benefit from expanded imaging capabilities and valuable product discounts.
Bio-Rad is also dedicated to providing personal customer service, including instrument and application support both before and after purchase. Our highly trained field application specialists and technical support staff are ready to help you with challenging applications. For more information or for assistance in selecting the appropriate imaging system and accessories for your advanced imaging needs, contact your local
Bio-Rad sales representative.
B
A
B
Table 2. Effect of scan resolution on file size.
Scan Size
10 x 10 cm
10 x 10 cm
10 x 10 cm
20 x 25 cm
35 x 43 cm
Resolution
100 µm
50 µm
10 µm
10 µm
10 µm
Scan Time
3 min
5 min
60 min
~4 hr
~7.5 hr
File Size
2 Mb
8 Mb
180 Mb
915 Mb
2,900 Mb
Fig. 2.
Comparable sensitivity of detection for SYBR Green I. A,
Molecular Imager FX Pro Plus; B, competitor system. The same gel was imaged on both systems.
Fig. 3.
Comparable sensitivity of detection for ethidium bromide. A,
Molecular Imager FX Pro Plus; B, competitor system. The same gel was imaged on both systems.
Pixels/Band
360
1,440
36,000
36,000
36,000
Table 3. Molecular Imager FX Pro Plus multiimager system specifications.
Excitation source
Optional external lasers
Blue external laser
Red external laser
Sample size
Phosphor screen sizes
Linear dynamic range
Digital resolution
Scan resolution
Dimensions
Standard emission filters
Optional emission filters
Weight
Operating system
20 mW 532 nm diode-pumped solid-state laser
15 mW 488 nm argon ion laser
10 mW 635 nm diode laser
35 x 43 cm
20 x 25 cm and 35 x 43 cm
5 orders of magnitude (1:100,000)
16-bit
User-selectable 50, 100, 200, and 800 µm
68 x 57 x 30 cm (D x W x H)
526 nm SP, 390 nm BP, 530 nm BP, 640 nm BP, 555 nm LP
605 nm BP, 690 nm BP
31 kg
Windows NT/98/2000 or Macintosh OS 9
Cy is a trademark of Amersham Biosciences. Macintosh is a trademark of Apple Computer. SYBR,
SYPRO, and Texas Red are trademarks of Molecular Probes, Inc. Windows and Windows NT are trademarks of Microsoft Corp.
Bio Radiations Volume 109 19

™
Fittings compatible with a wide variety of tubing sizes
1/4-28 flatbottom fitting
Fig. 1.
Flow path design features increase resolution.
Overview
Since its introduction, the BioFrac fraction collector has established itself as a reliable, easyto-use, and versatile fraction collector. Of interest to many laboratories is its enhanced capability of filling microplates and microtube cluster racks used in microplate-based assays.
Introduction
The BioFrac fraction collector is superior to other fraction collectors on the market for collection of samples into microplates and microtube cluster racks. Its design incorporates a rack adaptor that holds a wide variety of microplate formats, plumbing that minimizes the system volume, and sample collection patterns that simplify automated microplate analysis. These improvements make the BioFrac the ideal solution for collecting samples that will later be processed using multichannel pipets or automated laboratory equipment, or analyzed using microplate readers.
Applications that are enhanced by this functionality include a wide variety of microplatebased assays such as protein and DNA quantitation, ELISA assays, drug screening,
1/4-28 flatbottom fitting
Diverter valve mounts on either side of collector.
Collect
Common
Waste enzyme activity assays, and analysis of proteinprotein and protein-DNA interactions. Another efficient technique is the use of microplates and multichannel pipets for preparation and loading of fractions onto SDS-PAGE gels for purity analysis.
Fraction Collection for Microplate-Based
Techniques
The BioFrac is designed with microplate-based sample collection in mind. It accepts a wide variety of microplate formats including microtube cluster racks and 12-, 24-, 48-, and 96-well microplates in both regular and deep-well formats.
Furthermore, the BioFrac is designed to work with robotic compatible microplates and microtube cluster racks that adhere to the Society for
Biomolecular Screening (SBS) standards for microplates ( www.sbsonline.com
).
The BioFrac fraction collector can fill deepwell microplates as well as traditional microplates due to the adjustable drophead, which can fit microplates of any height. Deep-well microplates are a convenient storage format for purified samples. These plates can hold up to 10 ml depending on the plate format and height. They are easy to store, stackable, and more convenient to work with than collection tubes. Additionally,
96-well deep-well microplates facilitate removal of samples for analysis since they are compatible with both 8-well and 12-well multichannel pipets.
Flow Path Design
The BioFrac fraction collector has been designed to collect small-volume samples. Collection of the small volumes used with microplate-based techniques requires special attention to flow path design (Figure 1). In order to get the maximum resolution from your chromatography system, the post-column plumbing should be optimized.
Sample resolution is lost when molecules separated during the chromatographic process are remixed in the tubing or inline devices. The primary cause of lost resolution is diffusion and mixing due to nonlinear currents within the plumbing. Regions of the system that have large internal volumes, such as detector flow cells, pH probe flow cells, valves, or backpressure regulators
20 Bio Radiations Volume 109

Collect chromatography fractions into
96-well SBS-standard microplates
Transfer fraction aliquots into a standard 96-well microplate
Collect chromatography fractions into 10 ml deep-well microplates
Add gel loading buffer to prepare samples for electrophoresis
Add Bio-Rad’s protein assay dye reagent concentrate
Capture data with the Model
680 microplate reader
Visualize highresolution fraction purity results in a minimum of time
Load samples and run SDS-PAGE gels
(Criterion ™ 12+2 and 26-well comb precast gels are multichannel pipet compatible)
Determine sample concentrations with easy-to-use Microplate
Manager ® analysis software
Bio Radiations Volume 109 21

can contribute significantly to this problem. To compensate for these factors, the BioFrac fraction collector offers well-designed post-column plumbing options. The BioFrac diverter valve has a small internal volume (12 µl) that is 100% swept volume. There are no areas of dead volume that could lead to trapping and remixing of samples and impurities. In addition, the BioFrac diverter valve can be mounted on either side of the collector, and its position adjusted to minimize tubing length. A wide range of tubing sizes can also be used with the BioFrac because its diverter valve and fittings are compatible with tubing as small as 0.0025" (65 µm) ID, which is ideal for maintaining the separation and concentration of small samples collected at low flow rates.
Collection Patterns
The BioFrac fraction collector allows you to select the collection pattern for placing samples into your microplates. Many of the automated instruments used to process and analyze samples in microplates require that the samples be loaded in a specific pattern (usually by row or column).
The BioFrac can be easily programmed to collect microplate fractions in a row, column, or serpentine pattern.
Sample Identification
The advanced programming features (Figure 2) of the BioFrac fraction collector help identify the precise location of your sample. The volume of the tubing and inline devices between the detector and the drophead results in an offset between the displayed location of a peak on a chromatogram and its actual location in the fraction collector. This offset can be significant when collecting small fractions, as is often done in microplates. The BioFrac delay volume feature can be used to synchronize fraction advances with your detector signal. This ensures that the location of the fraction advance marks on the chromatogram correspond to the actual fraction advances at the fraction collector.
Conclusion
The BioFrac fraction collector is uniquely suited for microplate-based collection. Its ability to hold a wide variety of microplate formats and to perform sample collection by row and column ensures its compatibility with equipment used in downstream sample processing and analysis. The
BioFrac collector’s diverter valve and tubing options keep system volumes to a minimum and reduce loss of chromatographic resolution due to diffusion. These features make the BioFrac fraction collector the best choice for microplatebased fraction collection.
Fig. 2.
Programmable delay volume ensures peak synchronization with fraction advance marks on your chromatogram.
22 Bio Radiations Volume 109

Bio
™
Chromatography Systems
.
The BioFrac ™ fraction collector allows you unprecedented efficiency through its ability to support a wide variety of collection racks, collection programming, and system integration. Work smarter and diminish time delays with both off-the-shelf and unique custom racks, quick and easy method programming, and a wide variety of collection modes. The BioFrac updates your chromatography system to meet the ever-changing formats of today’s applications. Now, choose your collection methods based on your needs rather than the limits of your existing fraction collector. Its flexibility makes the BioFrac a logical choice.
Ready for a Change? Contact Us.
Find out more about the versatile BioFrac fraction collector by visiting our web site at discover.bio-rad.com
Compatible with a range of off-the-shelf collection racks
Visit us on the Web at discover.bio-rad.com
Call toll free at 1-800-4BIORAD (1-800-424-6723); outside the US, contact your local sales office.
Ice bath rack holds 4 microtiter tube trays, microplates, or test tubes
BioFrac with
Prep-20 adaptor

P
Proteomics collectively analyzes the proteins that are regulated, expressed, or modified in the cell under different conditions (Liebler 2002).
Because proteins have diverse structural, enzymatic, and regulatory roles in cells, methods that offer opportunities to study such a broad range of processes are in demand. Proteomics may be most advantageous for studies that can utilize 2-D gel electrophoresis to provide a visual map for comparison of protein expression at given times and under known conditions.
Examples include studies of:
• Characteristics and effects of disease
• Responses to experimental treatments such as drugs, diet, or environmental conditions
• Developmental processes such as cell and tissue differentiation, morphogenesis, and aging
• Studies of closely related organisms to better understand evolutionary relationships and mechanisms of pathogenesis
Proteomics can be divided into two main subcategories: expression proteomics, the study to identify specific targets and markers, and functional proteomics, the study to define structure, interaction, and function. Expression proteomics begins with comparison of gels, which is an excellent way to observe up/down regulation, on/off expression, and modification of proteins.
While expression proteomics can be viewed as the method of discovery — it allows identification of a protein of interest — functional proteomics allows more detailed analysis of this protein’s structure, role, cellular location, and interaction with other proteins.
For example, in a disease state such as cancer or diabetes, the normal expression of particular proteins has somehow changed, making them potential targets for investigation and, eventually, possible drug therapy. Comparison of normal protein expression with that of the diseased state may indicate differences in protein expression and thus identify potential target proteins.
However, just seeing differences in these 2-D gel comparisons does not necessarily implicate a protein as a candidate for a drug therapy approach.
Further study of the function of the proteins of interest can indicate their role in the disease process. Functional proteomics technologies can quantitate, localize, and observe proteins discovered during expression analysis to determine whether they are good candidate “target molecules”.
The entire process of discovering and determining the function of a protein via expression and functional proteomics is integral to developing a potential therapeutic approach for the treatment of a disease.
More sophisticated tools for protein experimentation and analysis are continually being developed, further improving proteomic technologies and opportunities for applications.
In addition, ever expanding collections of information are accessible through publications and databases. These new techniques and technologies require knowledge and expertise in a wide variety of disciplines; therefore, it is critical to have access to useful resources.
Bio-Rad offers the ProteomeWorks ™ system, which is a set of seamlessly integrated technologies for expression proteomics, and a variety of functional proteomics tools. Information about these tools can be found on the following pages.
Researchers at the Tokyo Metropolitan Institute of Gerontology use proteomics to look at the mechanisms of advanced aging caused by Werner’s syndrome (WS). WS is an autosomal recessive disorder that is responsible for the onset of premature aging.
Tosifusa Toda and colleagues have found that WS patients’ fibroblasts have a shorter lifespan compared to fibroblasts from healthy individuals. Using 2-D electrophoresis with PDQuest ™ imaging software, Toda et al. compared the proteomic profiles of fibroblasts of patients with WS and those in a healthy state grown in vitro. Their results show that the changes in protein patterns during aging in vitro do not necessarily correspond to the changes in aging WS fibroblasts, except for three proteins abundant in WS fibroblasts, which increase in abundance during aging in vitro. These results suggest that the premature aging process of WS fibroblasts shares only part of the aging process of normal fibroblasts.
For more information on this research, go to http://proteome.tmig.or.jp/2D/
Bio Radiations Volume 109 25

™
The ProteomeWorks system, developed jointly by
Bio-Rad Laboratories and Micromass, Inc., is an integrated platform that provides expression proteomics tools to meet the need for rapid and systematic protein identification. It provides a high-throughput protein analysis system with marked improvements in efficiency and workflow; technologies to separate, isolate, and identify proteins; reproducibility to generate meaningful data; and powerful bioinformatics tools to interpret and manage the complex data generated.
The following describes the ProteomeWorks system workflow and technologies that enable protein identification.
Sample preparation is the first step in any proteomics project, and the most critical in achieving reproducible and meaningful results
(Liebler 2002). The key to being able to identify low-abundance proteins in a complex mixture is enrichment of those proteins. The method of enrichment and protocol used depends on a variety of factors such as solubility, size, charge, and isoelectric point (pI) of the proteins being investigated.
The ProteomeWorks system provides a variety of methods for prefractionation of proteins from complex biological samples. The Rotofor ® preparative isoelectric focusing (IEF) system fractionates complex protein samples in a liquid solution where they are easily accessible for
2-D array generation. Another successful prefractionation strategy is to perform sequential
Bladder cancer includes a variety of tumor types. One type, squamous cell carcinoma
(SCC), has a morphology and protein expression profile similar to that of keratinocytes
(skin cells of the epidermis). Determining the progression of cancer depends on many factors such as nuclear polymorphism, nuclear to cytoplasmic ratio, chromatin clumping, etc. Since these observations are highly subjective, a more reliable way to objectively classify bladder SCC would help direct studies of molecular mechanisms underlying the cancer progression. Danish scientists are exploring the possibility of using proteome expression profiles of these carcinomas as fingerprints to identify tumor subtypes and define the degree of abnormality. Identification and characterization of SCC proteins has been advancing rapidly by comparing their expression profiles to 2-D PAGE proteomic databases of keratinocytes.
For more information on this research, go to http://proteomics.cancer.dk/ extractions with increasingly potent solubilization reagents such those in the ReadyPrep ™ sequential extraction kit. Affinity, ion exchange, and ceramic hydroxyapatite chromatography are also effective methods to prefractionate complex protein mixtures prior to 2-D separations. (Bio-Rad offers both prepacked chromatography columns and bulk media that can be run on instruments such as the BioLogic DuoFlow ™ system.) The desired protein fraction should be in a low-ionic-strength denaturing buffer that maintains the native charges of proteins and keeps them soluble for the next step, 2-D array generation (bulletin 2651).
2-D electrophoresis has become the standard proteomics separation technique, because it has the ability to resolve complex mixtures of thousands of proteins in a single gel.
Standardization of this technique with precast immobilized pH gradient (IPG) strips and SDS-
PAGE gels has simplified the procedure and provides improved gel-to-gel reproducibility.
First Dimension: IEF
Differences in proteins’ pI are the basis of separation by IEF (Garfin 2000). In practice, this means that proteins are applied to a gel and separated within an appropriate pH range using an electrophoresis cell such as the PROTEAN ® IEF cell. Immobilized pH gradient strips provide a convenient way of resolving proteins by pI
(Garfin 2000). ReadyStrip ™ IPG strips include a variety of pH ranges and gel lengths for different levels of resolution. Appropriate sample application and incubation time prior to focusing can help optimize protein resolution (Sanchez 1997).
Second Dimension: SDS-PAGE
The second dimension of protein separation, following IEF, usually consists of gel electrophoresis to separate the proteins according to their molecular weight (CancerWEB on-line medical dictionary). For the high-throughput needs of proteomics, Bio-Rad offers a variety of precast gels that hold different strip lengths, as well as the
Dodeca ™ family of electrophoresis cells, which accommodate up to 12 gels per run.
For proteomic applications, the goal of gel electrophoresis is to separate proteins within a mixture so that they can be identified. Proteins
26 Bio Radiations Volume 109

PROTEAN ®
ReadyStrip
IEF Cell and
™ IPG Strips
PROTEAN Plus ™
Dodeca ™ Cell www.ProteomeWorksSystem.com
TM
ProteomeWorks ™
Spot Cutter
M@LDI - HT ™
2-D Gel MS
Sample Preparation
Protein Prefractionation and Separation
2-D Electrophoretic
Separation
Stains, Buffers, Standards, and Blotting
Imaging Systems
Image Analysis
Automated Protein Excision
Automated Protein Digestion
Multi-Dimensional HPLC
MALDI and Electrospray
Mass Spectrometry
Global Bioinformatics
VersaDoc ™
Imaging System
PDQuest
ProteinLynx ™
™ and
Sotfware
™
™
™
ProteomeWorks is the global alliance between Bio-Rad Laboratories, Inc. and Micromass-Waters, Ltd., dedicated to furthering proteomics research.

Bio-Rad Imaging Tools
• GS-800 ™ calibrated densitometer:
For colorimetric stains (Coomassie
Blue, silver, copper, zinc) and
X-ray film
• VersaDoc ™ imaging system: For colorimetric stains (Coomassie
Blue, silver, copper, zinc) and
X-ray film
• Molecular Imager FX ™ multiimager systems: For fluorescent dyes
(SYPRO Orange, SYPRO Red,
SYPRO Ruby, FITC, Texas Red) and storage phosphor imaging of 35 S-labeled proteins
TM in gels can be made visible by staining them.
Recommended stains compatible with downstream proteomic applications, such as mass spectrometry and Edman sequencing, include the Silver Stain
Plus ™ stain, Bio-Safe ™ Coomassie stain, and
SYPRO Ruby protein gel stain (bulletin 2651).
The ProteomeWorks system provides a variety of instruments for imaging gels following staining or labeling, including densitometry for visual stains, combined multifluorescence and epiillumination for versatility, laser scanning for high-resolution fluorescence imaging, and storage phosphor imaging (see sidebar). After a digital image has been acquired, the next step is to identify proteins of interest.
One of the most common approaches in expression proteomics is to compare differences in the protein 2-D patterns of complex samples.
Computer-assisted image analysis software has made this process much easier and more efficient by allowing storage and structuring of large amounts of collected experimental image data, rapid and sophisticated analysis of information, and creation of 2-D protein databanks for comparison of data between different laboratories.
PDQuest ™ software is the core image analysis component for the ProteomeWorks system, with many functions and features that increase the accuracy and efficiency of protein pattern detection. PDQuest integrates the ProteomeWorks system; it can control all Bio-Rad imagers and the
ProteomeWorks spot cutter. It also allows transfer of mass spectrometry (MS) annotations from
Micromass MassLynx software. PDQuest software v. 7.1, the most recent version, has an automated gel matching feature for rapid, reproducible processing of experimental results. In addition, data from a few gels or thousands of gels can be analyzed by a wide variety of powerful analysis query tools. Significant changes can be identified using analysis sets, which can be built through several analytical tools: quantitative, qualitative, parametric and nonparametric statistical tests, and arbitrary and Boolean associations. In addition to spot comparison across different gels, the software can also annotate protein spots by linking to MS data identification software or by user input.
Image analysis yields quantitative and qualitative information about the proteins in a sample, and stores the information in files, to which annotations can be added. The analysis sets created in PDQuest software are used directly as the cut set for the ProteomeWorks spot cutter through the integrated excision tool. Alternatively, a point, click, and cut interface for the spot cutter is available through the basic excision tool.
Integration between the spot cutter and
PDQuest software allows “error-free” data tracking, reduces the time needed to extract the proteins manually, and significantly reduces the keratin contamination problem associated with manual processes. Excised protein spots are deposited into microtiter plates and are ready for further automated processing. PDQuest software tracks the protein spots through spot cutting and passes the information to the Micromass Mass PREP station and M@LDI HT 2-D gel-MS analyzer for final peptide identification.
The excised protein spots are destained, chemically modified, and digested on the Mass PREP station, a multifunctional robotic protein handling system, in preparation for identification by MS. Through advances in software control and bioinformatics, the whole process of data acquisition, data processing, and database searching has been fully automated.
The Micromass M@LDI HT is one of a new generation of networked 2-D gel-MS analyzers for high-throughput protein identification. M@LDI
HT is the primary MS data acquisition device of the ProteomeWorks system, and features a fully automated target plate autochanger for increased
28 Bio Radiations Volume 109

throughput. Integration with PDQuest 2-D analysis software enables automatic annotation of identification results on gel images. Networking allows distribution of data capture, protein assignment, and results presentation within a secure client-server architecture, taking maximum advantage of computing power.
Major advances in the field of molecular biology, coupled with advances in proteomic technologies, have led to an explosive growth in the amount of data generated by the scientific community. This deluge of information has, in turn, led to an absolute requirement for computerized databases to store, organize, and index the data, and a need for specialized tools to view and analyze the data.
WorksBase ™ software is the ProteomeWorks system’s bioinformatics platform. WorksBase allows entry and management of data generated from all steps of a proteomics project’s workflow, from reagents and their storage locations to sample preparation protocols, as well as image and
MS information. It is a relational database that integrates with the industry standard, Oracle 8 i , for handling and managing data. The system allows automation and integration of the collection and organization of data to increase throughput. The WorksBase platform allows seamless integration of image, laboratory, MS, and public or private reference data. In addition,
PDQuest integrates with WorksBase so that a user can store all information for a gel in a single platform.
Once the discovery phase of expression proteomics has been completed, the next step is to determine how the identified protein contributes to the function of a cell. In addition to protein purification tools such as chromatography media and systems,
Bio-Rad offers other proteomic tools that provide insight into the function of proteins of interest.
Confocal and multi-photon laser-scanning microscopy allow the acquisition of optical section images through fluorescent biological samples, where only light that is in focus is collected.
These techniques allow visualization of materials in three-dimensional tissue structures. The
Radiance2100 ™ alignment-free laser pointscanning systems utilize this principle to locate fluorescently tagged molecules of interest within tissues. The techniques can provide insight into the relationships of labeled proteins to other proteins, nucleic acids, and tissue structures, and how they change with time.
The Clonis ™ cellular laser observation and isolation system is a unique means of isolating cells for the purpose of protein enrichment or characterization. In addition to live cell isolation, it allows dissection of viable tissue sections, selective cell ablation, and micropatterning of cell growth surfaces. It allows viewing of the morphology, immunohistology, and growth patterns of cell and tissue samples.
The Bio-Plex ™ protein array system quantitates up to 100 different proteins and peptides in a single microtiter well, permitting the simultaneous acquisition of valuable information from rare or volume-limited samples. Assay kits for human and mouse cytokines and phosphoproteins are currently available. These target molecules can be used to help define complex relationships among proteins in signal transduction pathways.
Custom assays can be also developed for specific applications. This approach can be useful, for example, in pharmacological applications, when studying protein profiles helps to understand a drug’s mode of action and predict its toxicity.
These proteomic solutions provide a powerful and elegant means of pursuing many important research efforts. A potential protein of interest is identified through expression analysis and its role in a cellular or developmental process is inferred from functional analyses. Through such a proteomic approach, the effectiveness of therapeutic drugs and other treatments can also be evaluated. The combination of a variety of technologies allows the display, characterization, and quantitation of complex protein arrays. As these technologies advance and as new techniques are developed, they will enhance our fundamental understanding of biology and lead to new applications of our knowledge.
More information about Bio-Rad’s tools as well as proteomics conferences and links to other proteomics resources can be found at www.ProteomeWorksSystem.com
Software and Online
Resources
• Image acquisition, analysis, and spot excision: PDQuest 7.01
• Information management:
WorksBase 1.0
• Functional proteomics arrays:
Bio-Plex Manager ™ 2.0
• Confocal and laser scanning microscopy: LaserSharp ™ 2000
• Clonis system: Clonis software
• ProteomeWorks system web site:
ProteomeWorksSystem.com
• ExPASy molecular biology server: www.expasy.org/
References
Blackstock WP and Weir MP,
Proteomics: quantitative and physical mapping of cellular proteins, Trends Biotechnol 17,
121–127 (1999)
Garfin DE, Isoelectric focusing, pp 263–298 in Ahuja S (ed),
Separation Science and
Technology, Vol 2, Academic Press,
San Diego (2000); also available as
Bio-Rad bulletin RP0016
Liebler DC, Introduction to
Proteomics: Tools for the New
Biology, Humana Press, Inc.,
Totowa, MJ (2002)
Sanchez JC et al., Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients, Electrophoresis 18,
324–327 (1997)
On-line medical dictionary, http://cancerweb.ncl.ac.uk/omd/
2-D electrophoresis for proteomics:
A methods and product manual,
Bio-Rad bulletin 2651
Coomassie is a trademark of
Imperial Chemical Industries PLC.
ExPASy is a trademark of Institut
Suisse de Bioinformatique. M@LDI,
MassLynx, and Mass PREP are trademarks of Micromass, Inc.
SYPRO and Texas Red are trademarks of Molecular Probes, Inc.
Bio Radiations Volume 109 29

™
Cheryl S Aberin and Samuel G Franklin, PhD, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA
Fig. 1.
Purification of plasmid DNA on CHT II support.
Buffer A: 10 mM sodium phosphate
+ 1 mM EDTA, pH 7.0
Buffer B: 0.4 M sodium phosphate
+ 1 mM EDTA, pH 7.0
Flow rate: 1.5 ml/min
Gradient: 0–100% buffer B for 10 column volumes
Fraction size: 2.0 ml
Introduction
Plasmid DNA is being used successfully as a gene delivery vector in a variety of clinical applications (Smith et al. 1999). Plasmids for gene therapy are usually produced in an E. coli host. One of the technical challenges associated with producing plasmid DNA of gene therapy grade is the removal of contaminants such as bacterial chromosomal DNA, RNA, host proteins, and endotoxin.
Chromatography plays a key role in the largescale purification of plasmids, both as a process step and as an analytical tool. Different types of chromatography such as ion exchange, gel filtration, reverse phase, and affinity have been used for the separation of plasmid DNA.
Chromatography is often preceded by RNase treatment, diafiltration, precipitation, dilution, and other steps, which can increase process costs and decrease productivity and product recovery.
Thus, there is a significant need for an alternative plasmid purification method.
We have developed a very effective and rapid method for preparing plasmid DNA from E. coli that should be readily scalable. Use of our patented modified alkaline lysis procedure* allows direct loading of the clarified lysate to CHT ceramic hydroxyapatite, thus eliminating the need for any other sample preparation or handling steps.
1.0
2 6 10 14 18
Fraction number
22 26 30 34 38 42 46 50
100
0.8
1
0.6
0.4
3
2
0.2
0.0
0
0 30
Run time, min
60
*The plasmid purification process described here is covered by US patent 6,406,892.
Methods and Results
Cell Lysis
A plasmid DNA sample (5,955 base pairs, derived from pUC19) was grown in E. coli strain DH5 α .
Cells were grown in Terrific Broth medium supplemented with 100 µg/ml ampicillin. The cell pellets were resuspended in 40 mM sodium phosphate buffer, pH 8.0, containing 25 mM
EDTA in an ice bath. AquaPure ™ lysis buffer
(catalog #732-6541) was added to the suspension, mixed immediately, and stored on ice for 3–5 min.
Potassium chloride (1 ml, 3.0 M) was added immediately and mixed, and the resulting suspension was adjusted to pH 4.5 with 1.0 N
HCl, then neutralized to pH 7.0 with NaOH solution. The crude lysate was centrifuged at
15,000 x g for 20 min at 4°C. The precipitate was removed by filtering on a 70 µm nylon cell strainer.
Purification
The clear E. coli lysate was injected onto a CHT column (Econo-Pac ® CHT Type II, 5 ml cartridge, catalog #732-0081) with a particle size of 20 µm and a nominal pore diameter of 800–1,000 Å.
The column was washed with 5 column volumes of 10 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA, followed by a linear gradient to 0.4 M sodium phosphate buffer for
10 column volumes at a flow rate of 1.5 ml/min.
The elution process was monitored at 254 nm
(Figure 1). The fractions collected were analyzed spectrophotometrically at 260 and 280 nm to determine DNA and protein content.
DNA purity of fractions 35 to 37 (Figure 1, peak 3) was analyzed by electrophoresis in a 0.8% agarose gel (Figure 2, lanes 13–15). Fractions 35,
36, and 37 were found to contain pure plasmid
DNA and some of the nicked species. These fractions were digested with Eco RI restriction enzyme for 1.5 hr at 37°C. The digested DNA was analyzed by 0.8% agarose gel electrophoresis.
The gel results indicated that the supercoiled
DNA (undigested) migrated faster than the linearized (digested) DNA (Figure 3, lanes 6–11), confirming the identity and purity of the supercoiled plasmid DNA.
30 Bio Radiations Volume 109

Fractions from the CHT column were analyzed for nucleic acid content, genomic DNA, endotoxin, and protein. The results are in the table.
Plasmid Binding Capacity
The dynamic binding capacity of CHT was tested using purified plasmid DNA (pS3, 10 kb) and a
1.0 ml Econo-Pac cartridge. Using the buffer system described in Figure 1 at a flow rate of
0.5 ml/min, we loaded pure plasmid to saturation and determined recovery of the eluted DNA by spectroscopy at 260 nm. The recoverable dynamic binding capacity was 0.23 mg/ml, representing a recovery of 55.0%. Different plasmids and buffer systems may exhibit different binding capacities or recoveries.
Discussion
The method we devised for cell lysis coupled with chromatography on CHT was shown to provide pure plasmid DNA in a single step. The elimination of sample manipulation prior to loading the CHT column increases productivity and should result in improved recovery. Addition of a polishing or capture step to the process should further enhance the clearance of host cell contaminants.
Reference
Smith GJ III et al., Fast and accurate method for quantitating
E. coli host-cell DNA contamination in plasmid DNA preparations, Biotechniques 26, 518–526 (1999)
DH5
α is a trademark of Life Technologies.
For additional copies of this article, request bulletin 2731 on the reader response card.
Table. Analysis of CHT II fractions.
Sample Total DNA
(µg/ml) *
Volume
(ml)
Genomic DNA
(µg/ml) **
Endotoxin
(EU/µg) ***
Protein
(µg/ml) †
Clarified lysate (load)
Flowthrough
2025
617.5
5
16
33.01
undetectable
33.3
12.3
1,812
564
Peaks 1 and 2
Peak 3
29.28
18.67
26
20
1.18
1.45
5.51
0.15
40
9
*A
260
/A
280
; **PCR assay; ***LAL assay; † BCA assay
No RNA contamination (<0.1 µg/sample) was detected by agarose gel electrophoresis (data not shown).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1 2 3 4 5 6 7 8 9 10 11
Genomic DNA
Dimer
Open circular (nicked)
Supercoiled
RNA
Fig. 2.
Electrophoretic analysis of purified plasmid DNA. Lane 1, 1 kb molecular mass ruler; lane 2, control plasmid DNA; lane 3, clear lysate; lanes 4, 5, and 6, flowthrough fraction number 3, 5, and
7; lanes 7, 8, 9, and 10, peak 1; lanes 11 and 12, peak 2; lane 13, fraction 35; lane 14, fraction 36; lane 15, fraction 37.
Genomic DNA
Dimer
Open circular (nicked)
Supercoiled
RNA
Fig. 3.
Restriction enzyme analysis of purified plasmid DNA. Lane 1,
1 kb molecular ruler; lanes 2 and 3, undigested and digested plasmid
DNA (control); lanes 4 and 5, undigested and digested lysate; lanes 6 and 7, undigested and digested fraction 35; lanes 8 and 9, undigested and digested fraction
36; lanes 10 and 11, undigested and digested fraction 37.
Bio Radiations Volume 109 31

™
Kristina Lind and Mikael Kubista, TATAA Biocenter, Medicinaregatan 7A/B, 405 30 Göteborg, Sweden
Fig. 1.
Schematic of real-time immuno-PCR. Capture antibody is adsorbed to the surface of the wells in a microtiter plate. Detection antibody is tagged with a DNA marker through a biotinstreptavidin-biotin linkage.
Introduction
A technique for antigen detection, called immuno-PCR, was developed by Sano et al.
(1992). It combines the molecular recognition of antibodies with the high DNA amplification capability of PCR. The procedure is similar to conventional enzyme-linked immunosorbent assays (ELISA) but allows for more sensitive detection. Instead of an enzyme, a DNA molecule is linked to the detection antibody and serves as a template for PCR (Figure 1). The DNA molecule is amplified and the PCR product is measured by gel electrophoresis. An improvement of this method is to amplify the oligomer in a real-time
PCR instrument, thereby eliminating post-PCR analysis (Sims et al. 2000). Further, real-time PCR is extremely accurate and sensitive, which should make it possible to quantitate very low amounts of DNA-coupled detection antibody with high accuracy. Here we present early results on the development of real-time immuno-PCR for prostate specific antigen (PSA) using the iCycler iQ system. PSA is a well-known tumor marker for prostate cancer and is widely used to detect, stage, and monitor the disease.
Methods
Anti-PSA10 and anti-PSA66 from CanAg
Diagnostics were used as capture and detection antibodies, respectively, in the sandwich immuno-
PCR assay. Anti-PSA66 was biotinylated using biotinamido-caproate-N-hydroxysuccinimide ester. Biotinylated DNA was generated by
Biotinylated DNA
Biotinylated detection antibody
Capture antibody
Streptavidin
Antigen amplifying a 1,098 bp fragment of the gusA gene
( E. coli β -glucuronidase gene) with a 5'biotinylated forward primer (biotin-AACTATG-
CCGGAATCCATCG-3') and unmodified reverse primer (5'-ACATATCCAGCCATGCA-
CAC-3'), and was purified with the QIAquick
PCR purification kit (QIAGEN).
Real-time PCR was performed in the Bio-Rad iCycler iQ system. The primers used were
5'-GTTAGCCGGGCTGCACTC-3' and
5'-ACATATCCAGCCATGCACAC-3', which produced a 71 bp product. Reaction volumes were 25 µl, containing 1x PCR buffer (Sigma),
4 mM MgCl
2
(Sigma), 200 µM dNTPs (Sigma),
0.08 µg/µl BSA (Fermentas), 1 U JumpStart Taq polymerase (Sigma), 300 nM of each primer, and 0.5x SYBR Green I (Molecular Probes, Inc.)
Cycling conditions were 95°C for 3 min, followed by 40 cycles of 95°C for 20 sec, 60°C for 20 sec, and 72°C for 25 sec. The fluorescence data used for quantitation were collected at the end of each 72°C step.
Results and Discussion
The sandwich real-time immuno-PCR system was assembled in a standard PCR plate from Bio-Rad as shown in Figure 1. To each well, 50 µl anti-
PSA10 (10 µg/ml in 0.2 M phosphate buffer) was added and allowed to adsorb to the surface of the
PCR plate overnight. The wells were then washed three times with wash buffer 1 (0.154 M NaCl,
5 mM Tris, pH 7.75, 0.005% Tween 20, 0.1%
Germall II), and the surface was then blocked from further adsorption by incubating at room temperature overnight with blocking buffer
(50 mM Tris, pH 7.0, 6% D-sorbitiol, 0.1% BSA,
0.05 % NaN
3
). The wells were washed with wash buffer 1, followed by incubation at room temperature with 5 µl of PSA standards
(containing 2.4 x 10 6 to 2.8 x 10 9 molecules) and
20 µl Tris-HCl buffered salt solution containing
BSA for 1 hr (CanAg PSA EIA instructions).
The samples were then washed three times with wash buffer 1. Biotinylated anti-PSA66 was diluted to 0.92 µg/L with phosphate buffer containing BSA and immunoglobulins to reduce nonspecific adsorption (CanAg PSA EIA instructions). To each well, 25 µl of the diluted
32 Bio Radiations Volume 109

biotinylated anti-PSA66 was added and the samples were incubated for 1 hr at room temperature. After washing six times with wash buffer 1, the wells were incubated for 30 min at room temperature with 25 µl streptavidin solution
(0.3 µg/ml streptavidin in 10 mM Tris-HCl, pH
7.5, 50 mM KCl, 2.5 mM MgCl
2
). Thereafter, the samples were washed six times with wash buffer 1 and incubated with 25 µl of biotinylated DNA marker (15 pM). The wells were then washed six times with wash buffer 1, followed by four times with wash buffer 2 (0.154 M NaCl, 5 mM Tris, pH 7.75), and then left to stand in wash buffer 2 for 1 hr before removing the buffer. Finally, the
PCR mix was added to the wells and real-time
PCR was performed in the iCycler iQ system
(Figure 2). Approximately 15–20 cycles were needed to reach threshold, set at 10,000 relative fluorescence units (RFU), which was substantially above the background but still in the exponential growth phase of the PCR. The C
T vs. log
(concentration) plot was linear with a large correlation coefficient (r = 0.986), showing that the assay correctly and accurately reflects the amount of PSA in the studied range. However,
PCR products that appear in negative control samples limit sensitivity. Analysis revealed two sources of these products. One is formation of primer-dimers, which form independently of the presence of template. There are several ways to suppress their formation through assay optimization techniques. The second source of error is nonspecific adsorption of the detection antibody or DNA. This is common to standard ELISA and can be reduced by proper blocking of the surface or by binding the capture antibody more tightly to allow for more extensive washing. These approaches are being developed.
The results presented here and also those reported previously (Sims et al. 2000) are promising indications that applications of real-time PCR will expand to protein detection. A challenging future task will then be to quantitate the levels of both mRNA and the corresponding protein in only a few cells, or perhaps even a single cell, to study the correlation between gene and protein expression.
100,000
10,000
1,000
0 5 10 15 20 25 30 35 38
Cycle
Acknowledgements
We thank CanAg Diagnostics for supplying antibodies, antigens, and buffers. We also thank our colleagues at the TATAA Biocenter for valuable discussions.
References
CanAg PSA EIA Instructions, Product No. 300-10, June 2000.
Available from CanAg Diagnostics, www.canag.se
Sano T et al., Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates, Science 258,
120–122 (1992)
Sims PW et al., Immunopolymerase chain reaction using realtime polymerase chain reaction for detection, Anal Biochem
281, 230–232 (2000)
Practice of the patented polymerase chain reaction (PCR) process requires a license. The iCycler iQ system includes a licensed thermal cycler and may be used with PCR licenses available from Applied Biosystems. Its use with authorized reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Some applications may require licenses from other parties.
Germall is a trademark of ISP Chemicals, Inc. JumpStart is a trademark of Sigma-Aldrich Corporation. SYBR is a trademark of Molecular Probes, Inc. Tween is a trademark of ICI Americas, Inc.
For additional copies of this article, request bulletin 2805 on the reader response card.
Fig. 2.
Amplification plot of realtime immuno-PCR of PSA using anti-PSA10 and anti-PSA66 as capture and detection antibodies, respectively. Threshold was set to
10,000 RFU. Samples contained
2.8 x 10 9 , 9.4 x 10 8 , 9.4 x 10 7 ,
9.4 x 10 6 , and 2.4 x 10 6 molecules of PSA; negative controls were without PSA.
Correlation coefficient: 0.986 Slope: –2,209 Intercept: 23,591
Y = –2,209X + 23,591
Unknowns
Standards
25
20
15
5 6 7 8 9 10 log Starting quantity, number of molecules
Fig. 3.
C
T vs. log (concentration) plot. Samples shown in blue contain 2.4 x 10 6 to 2.8 x 10 9 molecules of PSA, while samples shown in red are negative controls without PSA.
Bio Radiations Volume 109 33

™
Giles Cunnick and Wen Guo Jiang, Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Cardiff, UK
Introduction
Lymphangiogenesis refers to the formation of new lymphatic vessels. This process may occur in normal developing tissues or in tumors. In addition to spreading by direct invasion and via the bloodstream, many malignant tumors, breast cancer for example, spread via the lymphatics once new connections have been established. For this reason, the biology of tumor lymphangiogenesis has important therapeutic implications. Methods to quantitate lymphangiogenesis have not previously been described. Measuring the density of lymphatic vessels has not been reliable due to a lack of sensitive lymphatic markers and antibodies.
Furthermore, it does not indicate the rate at which the production of these channels is occurring. A more accurate method would be to measure the level of mRNA for lymphatic markers in tissues, since this correlates with the rate of lymphatic synthesis.
Lymphangiogenesis has not been studied in detail, primarily due to the lack of specific markers for lymphatic endothelium. However, a novel and specific lymphatic endothelial marker has been described, called LYVE-1 (Banerji et al. 1999).
This is a surface epithelial receptor located on the lymph vessel wall. The receptor appears to be highly specific for lymph vessels and is completely absent from blood vessels (Skobe et al. 2001). By quantitating LYVE-1 mRNA in tissues, estimation of the rate of lymphangiogenesis in those tissues is possible. We report a new approach to enable the quantitation of LYVE-1 mRNA in breast cancer specimens using the iCycler iQ real-time PCR detection system.
Materials and Methods
Kits and Reagents
Primary human umbilical vessel endothelial cells from Clonetics were cultured in endothelial cell basal medium (also from Clonetics). The RNAzol
RNA extraction reagent, reverse transcription kits, and PCR master mix were purchased from
ABgene. Platinum supermix-UDG for quantitative PCR was purchased from Life
Technologies. The conventional PCR primers were designed by the authors and synthesized by
Life Technologies. The TOPO TA cloning kit from Invitrogen was used for cloning. The agarose gel extraction kit and plasmid extraction kit were purchased from QIAGEN Ltd. The ABI PRISM
BigDye terminator v 3.0 cycle sequencing kit with
AmpliTaq DNA polymerase, FS was purchased from Applied Biosystems. The TaqMan reagents for quantitative PCR of β -actin were purchased from Applied Biosystems.
RNA Extraction and cDNA Preparation
Thirty frozen archival breast cancer specimens were homogenized and the total RNA extracted using the standard RNAzol procedure. The concentration of RNA was measured with a spectrophotometer. Equal amounts of cDNA were subsequently synthesized in 20 µl reaction mixtures by reverse transcription. The cDNA from human umbilical vessel endothelial cells was also synthesized in a similar fashion.
PCR and Sequencing
A pair of primers specific for part of the LYVE-1 gene sequence was designed to yield a DNA product of 925 bp. Conventional PCR was performed using the cDNA of human umbilical vessel endothelial cells and normal breast tissue together with the PCR mix and LYVE-1F and
LYVE-1R primers to generate a 925 bp PCR product. The PCR product was sequenced to verify the LYVE-1 origin of the product.
LYVE-1 Cloning and Plasmid Preparation
The LYVE-1 PCR product was cloned using the pCR2.1-TOPO vector and One Shot E. coli , and the plasmid extracted from the E. coli using the plasmid mini purification kit. PCR was performed on the plasmid using the LYVE-1 primers to confirm that the isolated plasmid contained the correct DNA sequence. The concentration of the plasmid was determined by UV spectrophotometry. Following this, the concentration of the plasmid (copies/µl) was
34 Bio Radiations Volume 109

calculated using the known molecular weight of the plasmid plus insert. Serial dilutions of the plasmid were then made.
Quantitation of LYVE-1 in Breast Cancer Samples
The Scorpions probe/primers were formed by linking a forward primer and fluorescent probe using a PCR stopper (Oswel Research Products
Ltd) as described by Whitcombe et al. (1999) to generate a 103 bp product from both the LYVE-1 plasmid and cDNA. Using the iCycler iQ detection system, the plasmid standards and breast cancer cDNA were simultaneously assayed in duplicate 15 µl reactions as follows: uracil DNA glycosylase supermix (7.5 µl), forward primer/probe (0.3 µl), reverse primer (0.3 µl), plasmid or specimen cDNA (1 µl), water (5.9 µl).
PCR conditions were 95°C for 4 min, followed by
50 cycles of 95°C for 15 sec; 54°C for 20 sec;
60°C for 40 sec. Using purified plasmids as internal standards, the level of LYVE-1 cDNA
(copies/µl) derived from the breast cancer samples was calculated. The sizes of the PCR products were verified on agarose gels.
Results
Quantitative Real-Time PCR Using
In-House Standards
The standard curve generated by the LYVE-1 standards revealed a linear correlation between the log copy number of the purified LYVE-1 plasmid and the threshold cycle number, with a correlation coefficient of 0.987 (Figures 1 and 2).
Quantitation of LYVE-1 in Breast Cancer
LYVE-1 RNA was present in all 30 breast cancer specimens. The mean concentration of RNA in the specimens was 27.0 ± 12.6 copies/µl.
Discussion
Using a novel, specific lymphatic marker, LYVE-1, we were able to quantitate lymphangiogenesis using the iCycler iQ PCR detection system and
Scorpions-based probes. Using these systems, a highly sensitive and specific detection and quantitation of LYVE-1 was possible. This is the first time that it has been possible to quantitate lymphangiogenesis indirectly.
The use of known concentrations of plasmid standards containing the desired sequence enables the concentration of the sequence in the original tissue to be quantitated, provided that the levels of RNA are initially standardized. It was found that LYVE-1 existed in all tested breast cancer specimens. Using this method, comparisons may be made between different subtypes of breast carcinoma to see whether the rate of lymphangiogenesis is related to cancer subtype.
Other cancers may also be studied.
Fig. 1.
Real-time PCR of six different concentrations of LYVE-1 plasmid standards (10
6
, 10
5
, 10
4
,
10 3 , 10 2 , and 10 copies/µl), each performed 5 times.
2,000
1,600
1,200
800
400
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
Cycle
Correlation coefficient: 0.987 Slope: –3,340
Intercept: 37,242 Y = –3,340 X + 37,242
Unknowns
Standards
35
30
25
20
15
10
1 2 3 4 5 6 7 log Starting quantity, copy number
Fig. 2.
PCR threshold cycle number vs. log plasmid copy number.
References
Banerji S et al., LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan,
J Cell Biol 144, 789–801 (1999)
Skobe M et al., Induction of tumor lymphangiogenesis by
VEGF-C promotes breast cancer metastasis, Nat Med 7,
192–198 (2001)
Whitcombe D et al., Detection of PCR products using selfprobing amplicons and fluorescence, Nat Biotechnol 17,
804–807 (1999)
ABI PRISM and BigDye are trademarks of Applied Biosystems.
AmpliTaq and TaqMan are trademarks of Roche Molecular
Systems, Inc. One Shot, pCR, Platinum, and TOPO are trademarks of Invitrogen Corp. RNAzol is a trademark of
Cinna Scientific, Inc. Scorpions is a trademark of AstraZeneca
UK Ltd.
Practice of the patented polymerase chain reaction (PCR) process requires a license. The iCycler iQ system includes a licensed thermal cycler and may be used with PCR licenses available from Applied Biosystems. Its use with authorized reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Some applications may require licenses from other parties.
For additional copies of this article, request bulletin 2806 on the reader response card.
Bio Radiations Volume 109 35

®
Andrew Conn, Lindy Durrant, and Ian Spendlove, Cancer Research Campaign Academic Unit, Nottingham City Hospital, Hucknall Road, Nottingham NG5 1PB, UK
Introduction
Gene gun immunization through the skin is a reliable and reproducible method of DNA vaccine delivery, and has been shown to be capable of inducing protective immunity in animal models to both infectious diseases and cancer (Chen et al.
2000, Chen et al. 1999). Delivery of DNA using the gene gun is a highly efficient method of achieving antigen presentation, and, as a result, immunizations require 250–2,500 times less DNA than standard intramuscular delivery (Fynan et al.
1993). This is due to the dense network of
Langerhans cells that are found in the epidermis, acting as a source of antigen-presenting cells.
Bio-Rad’s Helios gene gun system delivers
DNA to the epidermis using helium-driven bombardment of DNA-coated gold microparticles.
The nature of immune responses generated following vaccination with DNA depends on a number of key factors, such as the route, method, and vaccination schedule employed. We have therefore performed a series of pilot experiments with a DNA plasmid that encodes the hepatitis B surface antigen (HBsAg) to investigate the use of the Helios gene gun system in a Balb/c mouse model. A comparison has been made between delivery of this plasmid by gene gun and by intramuscular injection.
Methods
Plasmids
The pCMV-S plasmid encoding the HBsAg subtype ayw was kindly provided by Dr Robert
Whalen, Maxygen, USA. DNA was prepared using the QIAGEN EndoFree Plasmid Mega kit.
The presence of the HBsAg insert was verified by restriction enzyme digestion using Bam HI and analysis by agarose gel electrophoresis.
Preparation of DNA-Coated Gold Microcarriers
On the day prior to vaccination, pCMV-S plasmid
DNA was precipitated onto gold microcarriers as detailed in the Helios gene gun system instruction manual. Briefly, 8.3 mg of 1 µm gold microcarriers was resuspended by sonication in 100 µl of 0.05 M spermidine. Eighteen micrograms (18 µg) of DNA at a concentration of 1 mg/ml in endotoxin-free water was then added and sonicated; 100 µl of 1 M
CaCl
2 was then added dropwise. This gold-DNA mixture was allowed to stand for 10 min before being washed 3 times in 250 µl of 100% ethanol.
After the final wash, the pellet was resuspended in
200 µl of 0.025 mg/ml polyvinylpyrrolidone (PVP) in 100% ethanol, transferred to a 15 ml tube, and made up to 1 ml with PVP/ethanol. This resulted in a microcarrier loading quantity (MLQ) of 0.5
mg of gold per shot and a DNA loading ratio
(DLR) of 2 µg/mg gold, which results in the delivery of 1 µg of DNA per shot.
Loading DNA/Microcarrier Suspension Into
GoldCoat ™ Tubing
This was performed as detailed in the Helios gene gun system instruction manual, with 1 ml of
DNA/microcarrier suspension being used to produce
17 coated 0.5-inch cartridges, which were then stored overnight at 4°C with desiccant prior to use.
Vaccination of Mice
Female Balb/c strain mice aged 6–8 weeks were obtained from Charles River, UK, and housed at the Biomedical Services Unit, University of
Nottingham, UK.
For gene gun delivery, the abdominal fur of each mouse was removed with electric clippers prior to each vaccination. The barrel liner of the
Helios gene gun was then held directly against the abdominal skin, and a single DNA/microcarrier shot delivered using a helium pressure of 400 psi.
For intramuscular delivery, 100 µg of pCMV-S
DNA in endotoxin-free water with 0.2 µM CpG oligonucleotide was administered into the quadriceps muscle using a 1 ml insulin syringe.
Mice were not anesthetized and the muscle was not pretreated prior to vaccination.
36 Bio Radiations Volume 109

Vaccination Protocols
Two separate experiments were undertaken to assess DNA delivery by the Helios gene gun.
In the first experiment, one or two vaccinations by gene gun delivery were compared to the intramuscular route. Two groups of six mice received one pCMV-S DNA vaccination by either gene gun or intramuscular delivery.
Another two groups of six mice received one pCMV-S DNA vaccination each at weeks 0 and
2 by either gene gun or intramuscular delivery.
Antisera were then obtained at week 4 by postmortem cardiac puncture.
In the second experiment, the duration and degree of antibody response generated following two gene gun vaccinations was investigated. Six mice received one pCMV-S vaccination each by gene gun at weeks 0 and 2, and then antisera were obtained by tail bleeds at weeks 4, 6, 9, and 12 after vaccination.
ELISA to Show Antibody Response to pCMV-S
DNA Vaccination
ELISA was performed as described by Davis et al.
(1996). Microplates (96-well) were coated with
100 µl of 1 µg/ml HBsAg subtype ayw (Rhein
Biotech, Dusseldorf, Germany) and stored at 4°C overnight. Plates were then washed twice in phosphate buffered saline (PBS) containing 0.1%
Tween 20, and blocked with 200 µl of 10% fetal calf serum (FCS) in carbonate/bicarbonate buffer pH 9.6 (0.159 g Na
2
CO
3 and 0.293 g NaHCO
3
100 ml distilled water). Tenfold serial dilutions in
(1:10 to 1:10,000) of antisera from the immunized mice were made in PBS, 10% FCS, 0.05% Tween
20. After the plates were washed again five times in the previous wash solution, 100 µl of each serial dilution was added to the wells, and the plates were incubated at room temperature for 1 hr.
Following five more washes, 100 µl of 1:1,000 rabbit anti-mouse-HRP (Serotec Ltd, Oxford,
UK) in PBS, 10% FCS, 0.05% Tween 20 was added to each well and the plates incubated at room temperature for 1 hr. Plates were then washed five times before adding 150 µl of ABTS substrate solution and reading the absorbance at 405 nm.
ELISA to Show Antibody Subclass Following pCMV-S DNA Vaccination
ELISA was performed as described above with the exception that the rabbit anti-mouse HRP antibody was replaced with 100 µl of 1:1,000 goat anti-mouse IgG
1
-HRP or 1:1,000 goat anti-mouse
IgG
2a
-HRP antibodies (Serotec Ltd) in order to detect the subclass of antibody response generated.
To determine the relative affinities of the anti-
IgG
1 and anti-IgG
2a antibodies, a 96-well plate was coated with 50 µl of 1:1,000 rabbit anti-mouse immunoglobulins (Dako, Ely, UK) and stored at
4°C overnight. Fifty microliters (50 µl) of the control IgG
1 antibody 730 and IgG
2a antibody
1143B7 were then added at concentrations of 10,
3, 1, 0.3, 0.1, 0.03, and 0.01 µg/ml and the plates incubated at room temperature for 1 hr. Next, 50
µl of 1:1,000 rabbit anti-mouse-HRP antibody was added and the plates incubated for 1 hr at room temperature. ABTS substrate solution (150 µl) was then added and absorbance read at 405 nm.
Results
Intramuscular Vaccination
None of the six mice that received a single intramuscular vaccination developed an antibody response to the HBsAg. However, a response was obtained in four of six mice (detected at a 1:10 antiserum dilution) following two intramuscular vaccinations (Figure 1).
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1:10 1:100 1:1,000
Antiserum dilution
1:10,000
Fig. 1.
ELISA to show the presence of antibodies against
HBsAg following two intramuscular vaccinations with pCMV-S ( ■ ).
Control sera from unimmunized mouse in green ( ■ ). Error bars indicate standard deviation.
Bio Radiations Volume 109 37

Fig. 2.
ELISA to show the presence of antibodies against
HBsAg following Helios gene gun vaccination with pCMV-S. Single
DNA vaccination ( ● ), two DNA vaccinations ( ■ ), and control sera from unimmunized mouse ( ■ ).
Error bars indicate standard deviation.
Fig. 3.
ELISA to show the IgG antibody subclasses following pCMV-S vaccination by intramuscular and gene gun delivery.
IgG
1 subclass in green ( ■ ), and
IgG
2a subclass in gray ( ■ ). Error bars indicate standard deviation.
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1:10
Gene Gun Vaccination
Four of six mice that received a single Helios gene gun vaccination developed an antibody response to the HBsAg (detected at a 1:10 antiserum dilution), with one of these responses measured at a dilution of 1:100. All six mice that received two gene gun vaccinations demonstrated antibody responses, detectable at an antiserum dilution of
1:10,000 for two mice, 1:1,000 dilution for three mice, and 1:100 dilution for one mouse, respectively (Figure 2).
Duration of Antibody Response
Antisera obtained at weeks 4, 6, 9, and 12 after gene gun vaccination were all assessed by ELISA for the presence of antibodies to HBsAg. At all time points there was a significantly higher antibody titer compared to unimmunized control sera at a dilution of 1:1,000 (Figure 4). Antibody titers for the week
12 antisera are shown in Figure 5.
1:100 1:1,000
Antiserum dilution
Antibody Response by Subclass
1:10,000
The relative binding activity of the anti-IgG
1 antibody was greater than that of the anti-IgG
2a antibody. After taking this into account, we found that intramuscular vaccination with pCMV-S resulted in a predominantly IgG
2a response, while Helios gene gun vaccination resulted in a predominantly IgG
1 response (Figure 3).
Intramuscular Gene gun
Route of DNA delivery
Unimmunized
Discussion
Delivery of the pCMV-S plasmid using the
Helios gene gun system was a simple and efficient method of DNA vaccination in Balb/c mice.
In these pilot experiments, gene gun delivery consistently produced a higher antibody response rate to vaccination than the intramuscular route.
A single gene gun vaccination resulted in an antibody response against the antigen (measured at
1:10 dilution of antisera) in 66% (four out of six) of mice, this being equal to the response frequency observed with two intramuscular vaccinations.
Following two gene gun vaccinations, all mice developed prolonged antibody titers that were detectable at 1:1,000 antiserum dilutions and had not declined at week 12. In contrast, two intramuscular vaccinations resulted in at least
100-fold lower titers of antibody, and in only 66%
(four out of six) of mice.
The isotype of an antibody is often a good indication of the direction in which the immune response has developed. The IgG antibody subclass produced in response to intramuscular pCMV-S delivery was predominantly IgG
2a
. This is indicative of the T helper class 1 pathway
(Th1). With Helios gene gun delivery of pCMV-S, the subclass produced tended towards IgG1, suggesting an antibody-based immune response mediated by the T helper class 2 pathway (Th2).
This is consistent with previous experiments using the pCMV-S plasmid (McCluskie et al. 1999).
The bias toward a Th1 or Th2 response following
DNA gene gun delivery appears to vary according to different reports and may be a consequence of the encoded antigen and the makeup of the
DNA construct.
This pilot study has allowed us to compare intramuscular vs. gene gun-mediated, intradermal immunization. It has demonstrated the reliability and ease of use of biolistic delivery, as well as the ability of biolistic delivery to generate specific long-lived immune responses. Future work will further elucidate the diversity of the immune responses that are generated following DNA
38 Bio Radiations Volume 109

delivery of the pCMV-S plasmid using the Helios gene gun system. In particular, future work will investigate whether pCMV-S vaccination by gene gun is able to stimulate cell-mediated immune responses in addition to the humoral responses demonstrated in our work to date. The
IPQSLDSWWTSL peptide containing the dodecameric class I L d
-restricted epitope of the hepatitis B envelope will be used in cytotoxic T cell assays and tetramer analysis. This not only allows the generation of standardized assays, but also provides a control against which other vaccines can be tested.
Conclusion
Delivery of the pCMV-S plasmid using the Helios gene gun produced titers of antibodies against
HBsAg that were up to 1,000 times greater than those observed with intramuscular vaccination.
Antibody responses following gene gun delivery of pCMV-S were long-lived and did not decline for at least 12 weeks after initial vaccination. The
Helios gene gun system represents a reliable and reproducible method of DNA vaccination, which, when used to deliver the pCMV-S plasmid, results in sustained high antibody titers.
References
Chen CH et al., Gene gun-mediated DNA vaccination induces antitumor immunity against human papillomavirus type 16 E7expressing murine tumor metastases in the liver and lungs,
Gene Ther 6, 1972–1981 (1999)
Chen Z et al., Cross-protection against a lethal influenza virus infection by DNA vaccine to neuraminidase, Vaccine 18,
3214–3222 (2000)
Davis HL et al., DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines, Proc Natl Acad Sci USA 93, 7213–7218 (1996)
Fynan EF et al., DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations, Proc Natl
Acad Sci USA 90, 11478–11482 (1993)
McCluskie MJ et al., Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates, Mol Med 5, 287–300 (1999)
Tween is a trademark of ICI Americas, Inc.
For additional copies of this article, request bulletin 2726 on the reader response card.
2.0
1.6
1.2
0.8
0.4
0.0
1:10
1.2
1.0
0.8
0.6
0.4
0.2
0.0
4 6 9
Weeks after first immunization
12
1:100 1:1,000
Antiserum dilution
1:10,000
Fig. 4.
Antibody titers against
HBsAg at a serum dilution of
1:1,000 at weeks 4, 6, 9, and
12 after vaccination. pCMV-S vaccination in green ( ■ ), and unimmunized control in gray ( ■ ).
Error bars indicate standard deviation.
Fig. 5.
ELISA performed at week 12 to show the presence of antibodies against HBsAg following two Helios gene gun vaccinations with pCMV-S
( ■ ). Control sera from unimmunized mouse in green ( ■ ). Error bars indicate standard deviation.
Bio Radiations Volume 109 39

tips
™
™
High Throughput for Large 2-D Gels
The PROTEAN Plus Dodeca cell is a highthroughput, large format electrophoresis cell designed for 2-D applications. It can run up to twelve 25 x 20.5 cm (or 20 x 20.5 cm) gels that accommodate 24 cm IPG strips, matching the capacity of the PROTEAN ® IEF cell for optimal throughput. The PROTEAN Plus Dodeca cell is an integral part of the 2-D electrophoretic separation step in the ProteomeWorks ™ system.
Efficient Cooling for Reproducible Results
The PROTEAN Plus Dodeca cell requires a refrigerated circulator for optimal results because it is important to control the heat generated during long run times. An external refrigerated circulator* combines with the patent-pending
PROTEAN Plus Dodeca cell’s buffer recirculation pathway (BRP) to provide the cooling capacity required to achieve high-quality, reproducible results. Recirculation of the buffer throughout the cell maintains uniform buffer temperature for gelto-gel reproducibility (see Figure 1 for the correct setup of the BRP).
Keys to a Functional BRP
• The BRP is set up correctly when the buffer is pulled up through the lid, down through the pump, and back into the bottom of the tank (Figure 1)
• Set the buffer level just below the height of the plates (Figure 2). The buffer level will vary with different plate sizes
• The black fittings in the lid or the manifold tubing must only extend to just below the surface of the buffer level (approximately
1.0–1.5 cm) to provide optimum circulation
(Figure 2). If they extend further down into the running buffer, the buffer above them will not be sufficiently recirculated and the results may be adversely affected
• Always set the buffer recirculation pump to the maximum setting
Buffer exhaust tubing
Fig. 1.
Schematic diagram of the
BRP. The blue line and arrows represent the correct flow for buffer recirculation. The correct direction of flow is essential for successful operation of the BRP and for maintaining the buffer temperature.
Black fittings
Manifold
Manifold tubing
Quick connect fitting
Buffer recirculation pump
Ceramic cooling coils
40 Bio Radiations Volume 109

PROTEAN Plus Dodeca cell lid PROTEAN Plus Dodeca cell lid with manifold tubing attached
Buffer level
PROTEAN Plus hinged spacer plates PROTEAN II Ready Gel precast gels and
PROTEAN II handcast plates
When Is the Manifold Tubing Used and
Why Is It Important?
The manifold tubing is part of the BRP and is included with the cell. It is only required when running gels in the shorter plates (<26.5 cm wide), including PROTEAN II Ready Gel ® precast gel cassettes or any of the PROTEAN II glass plates.
The manifold tubing adds to the length of the black fittings in the lid to reach the buffer level, allowing the running buffer to be recirculated properly around these shorter plates. Without the manifold tubing, the buffer will not be recirculated.
PROTEAN Plus hinged spacer plates do not require the manifold tubing, because the plates are approximately 26.8 cm wide and the black fittings alone extend past the height of the plates.
Constant Voltage Run Conditions for
Consistent Runs
The design of the PROTEAN Plus Dodeca cell is unlike that of conventional electrophoresis cells, which electrically isolate the upper and lower buffer chambers. There is some current leakage built into this design, because the gels are immersed in a continuous buffer system and are supported by the gasket assemblies. These gasket assemblies create alternative pathways for current to flow besides through the gels. Thus, running at constant current can give variable results and is not recommended.
The PROTEAN Plus Dodeca cell gives the best results when it is run at constant voltage.
Consequently, the run times are more consistent and the results are more reliable. When the
Dodeca cell is running under constant voltage conditions, the initial current is high. However, the alternative pathways prevent the high current from traveling exclusively through the gels, and therefore the gels are not damaged. In addition, the cooling system is robust and constructed to manage any heat generated by the high current.
This design is ideal for high-throughput laboratories that demand simplicity and reliability.
The PROTEAN Plus Dodeca cell provides one of the simplest setup procedures for running large format gels. Simply slide the gel cassettes into the tank and you’re ready to start the run. You don't have to worry about upper buffer leaking to the lower chamber and spoiling a run.
For more information, visit us on the Web at discover.bio-rad.com
, request bulletin 2621
(product brochure), and see pages 150–151 of the
2002/2003 Life Science catalog.
* Purchase the refrigerated circulator separately. Recommended chillers include the Thermo NESLAB Models RTE-111 and
RTE-101, and the Thermo Haake Model WKL 26. Important specifications to consider are flow rate (about 4–10 L/min) and cooling capacity (minimum of 240 W at 20ºC).
Fig. 2.
Schematic diagram illustrating the proper use of the manifold tubing.
Bio Radiations Volume 109 41

tips
Proteins can be transferred from gels to membranes by methods such as capillary blotting, diffusion blotting, and electrophoretic transfer. Electrophoretic transfer is by far the most widely used method because of its speed and precision. There are two main types of electrophoretic blotting apparatus and transfer procedures: wet transfer, where a gel in contact with a membrane is submerged in transfer buffer in tanks (Figure 1), and semi-dry transfer, where the gel and membrane are first sandwiched between bufferwetted filter papers and then between flat-plate electrodes (Figure 2).
In principle, the wet and semi-dry transfer methods are the same. Gels and membranes are placed together in a filter paper sandwich between two electrodes. Proteins migrate to the membrane in response to a current (I) that is generated by applying a voltage (V) across the electrodes, following Ohm’s law (V = I x R). Resistance (R) is generated by the materials placed between the electrodes (i.e., the transfer buffer, gels, membranes, filter papers). An unfortunate byproduct of the transfer process, and one that must controlled, is heat. Heat generated during transfer may cause changes in the resistance of the system, which may lead to inconsistent field strength and transfer, or may cause the transfer buffer to break down or the gel to melt and stick to the membrane. Electrophoretic transfer systems are often limited by their capacity to dissipate heat.
Wet Transfer Using the Trans-Blot ® or Mini Trans-Blot ® Cells and the
Criterion ™ Blotter
Wet transfer systems are entirely submerged in transfer buffer. Although the significant volume of buffer enhances both the buffering and cooling capacity of wet transfer systems, additional cooling systems are offered by the Trans-Blot or Mini
Trans-Blot cells and Criterion blotter. These additional cooling systems prevent overheating during long, high-intensity, or native protein transfers. For most general-purpose and routine protein work, wet transfer systems are recommended for efficient and quantitative protein transfers.
The Trans-Blot, Mini Trans-Blot, and Criterion blotters differ primarily in the number and sizes of gels they can accommodate and in the flexibility of electrode positions. The Trans-Blot cell is generally used for large gels, although the gel holder cassettes can accommodate multiple smaller gels as well. Variable power settings can be used, from 30 V for overnight transfers to 200 V for rapid, 1-hour transfers. In addition, the Trans-
Blot cell’s electrode cards can be moved closer together to maximize field strength for highintensity applications.
Bio-Rad’s line of western blotting equipment. Left, the Trans-Blot ® SD semi-dry transfer system. Right, from back to front, the Trans-Blot cell, Criterion ™ blotter, and
Mini Trans-Blot ® cell.
42 Bio Radiations Volume 109

The Mini Trans-Blot cell and Criterion blotter accommodate small gels for rapid, high-quality blotting. The Mini Trans-Blot cell can transfer up to two mini gels (7.5 x 10 cm) in an hour and is available either as a complete blotting apparatus, or as a module that uses the buffer tank and lid of the Mini-PROTEAN ® 3 cell. The Criterion blotter can transfer up to two Criterion gels or four mini gels in 30–60 minutes.
Semi-Dry Transfer Using the
Trans-Blot SD Cell
In semi-dry blotting, the gel and membrane are sandwiched horizontally between two stacks of wetted filter papers that are in direct contact with closely spaced plate electrodes. The term “semi-dry” refers to the relatively small amount of buffer used, which is confined to the wetted filter paper.
The Trans-Blot SD semi-dry cell provides the maximum field strength for transfer because the electrode distance is determined by the thickness of the gel, membrane, and filter paper. Because the amount of buffer is limited to the filter papers, the cooling and buffering capacity of this system are limited. Therefore, this transfer system is used for rapid, high-intensity transfers and is best suited for transfer of mid-range proteins (10–100 kD); smaller proteins may pass through the membrane under high-intensity conditions and larger proteins may not have enough time to transfer.
The semi-dry method is favored in laboratories that run large numbers of blots because it is quick, requires less buffer, and is easier to set up than the wet transfer units. The Trans-Blot SD cell allows transfer in 15–30 minutes of large gels or multiple smaller gels that are placed side by side.
Ensuring Good Results
Bio-Rad has been manufacturing equipment and reagents for protein blotting for over 15 years and has specialized in-house expertise to help with blotting and detection questions. The most commonly asked questions are answered in the instruction manuals for the transfer units, in bulletin 1529, “Western Blotting Troubleshooter”, and in our searchable database of frequently asked questions (FAQs) available at discover.bio-rad.com
.
For more information about Bio-Rad’s blotting systems, consult the Guide to Blotting Cells on page 175 of the 2002/2003 catalog.
2
3
4
4
1
5
1
2
5
6
3
4
Fig. 1. Wet transfer system.
The gel holder cassette (1) holds the gel (2) and membrane (3) while fiber pads and filter paper (4) on both sides provide complete contact within the gel sandwich.
The gel cassette is inserted vertically into the buffer tank (5).
Fig. 2. Semi-dry transfer system.
Exploded view of (1) safety lid;
(2) cathode assembly with latches;
(3) filter paper; (4) gel; (5) membrane;
(6) filter paper; (7) anode platform;
(8) power cables; (9) base.
7
8
9
Bio Radiations Volume 109 43

@ the
Visitors to discover.bio-rad.com
will see a new home page with a cleaner look and more immediate access to information. Contact details are displayed right up front, local and major worldwide events are spotlighted, you get one-click access to job opportunities, and a “what's new” column highlights
Bio-Rad’s latest services, products, and other news of interest.
Bio-Rad is pleased to announce a new web site for our customers in Japan. This site offers answers to frequently asked technical questions and online access to literature in Japanese. In addition, you’ll find a list of local events, what’s new, employment opportunities, and product promotions tailored for you.
To access the site, from discover.bio-rad.com, select “Japan” from the country selection bar, or go directly to www.bio-rad.co.jp
and select the Life Science Research tab.
new
Gene Transfer Literature
Gene Pulser Xcell ™ Electroporation System Brochure
The new Gene Pulser Xcell is a versatile, modular electroporation system that delivers exponential or square waves. With manual and optimize capabilities, an intuitive interface, and preset programs, the
Gene Pulser Xcell provides power and reliability.
Request bulletin 2750 on the reader response card.
MicroPulser ™ Electroporator Brochure
For routine, high-throughput bacterial or fungal applications, the MicroPulser provides simple, efficient, and reproducible gene delivery.
Request bulletin 2751 on the reader response card.
XenoWorks ™ Microinjection System Brochure
The XenoWorks microinjection system is a complete line of instruments for micromanipulation and microinjection. The system features ergonomic height-adjustable joystick controls, micromanipulator position memories, and variable movement radius
(for more information, see page 14).
Request bulletin 2813 on the reader response card.
Gene Transfer Folder
The above brochures can be sent to you in our
Gene Transfer folder. Featuring a decision matrix to help you decide which method will best suit your needs, the folder is a convenient way to keep all of the brochures together.
Request bulletin 2749 on the reader response card.
Cytofectene ™ Transfection Reagent Brochure
Cytofectene is a powerful, ready-to-use cationic lipid transfection reagent that provides the highest transformation efficiencies for many cell types.
Request bulletin 2747 on the reader response card.
Bio Radiations Volume 109 45

new
Amplification Literature iTaq ™ DNA Polymerase and Core Reagents Flier iTaq DNA polymerase is a hot-start DNA polymerase suitable for many conventional PCR applications, and is qualified for use with the iCycler ™ thermal cycler.
The hot-start attribute is mediated through a specific antibody, and the enzyme is activated after an initial 3 minute denaturation step at 95°C. iTaq DNA polymerase ensures ease of use and high specificity.
Request bulletin 2779 on the reader response card.
iQ ™ Supermix Flier
Bio-Rad’s new iQ supermix is specially formulated to help you achieve unrivaled results in real-time PCR applications. It is qualified for use with the iCycler iQ ™ real-time detection system. This supermix will accelerate your research, facilitating the optimization of your reactions in the shortest time possible.
Request bulletin 2764 on the reader response card.
Beacon Designer Software Product Information Sheet
If you use the iCycler iQ ™ real-time PCR detection system, you can now take advantage of this comprehensive easy-to-use software package for realtime PCR assay design. Beacon Designer software from
PREMIER Biosoft Intl. is a probe and primer design package that makes designing probes effortless for important applications such as multiplex gene expression analysis and allelic discrimination. Beacon Designer designs optimal TaqMan and molecular beacon probes and primers.
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Electrophoresis Literature
Certified ™ Agarose Brochure
Certified agarose powders offer an agarose for every research need. These powders are genetic quality tested (GQT) grade to guarantee product quality in both routine separations and downstream molecular biology applications. This brochure includes detailed descriptions, gel images, analytical specifications, and functional tests for each type of agarose to help you select the best one for your application.
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Advantages of ReadyAgarose ™ Precast Gels Over 96-Well
Handcast Gels for High-Throughput Analysis
This tech note highlights the features and benefits of ReadyAgarose gels over handcast gels for highthroughput analysis. Learn about the convenience, time savings, and superior images obtained with these gels.
Request bulletin 2810 on the reader response card.
46 Bio Radiations Volume 109
Precision Plus Protein ™ Standards Brochure
This brochure gives you an overview of the features and benefits of the new Precision Plus Protein standards, with illustrations that show what the bands look like on gels and western blots. Precision Plus
Protein standards offer unsurpassed band sharpness, accurate molecular weight determinations, and lot-to-lot consistency. They are available in unstained, all blue, and dual color options, providing three ways to get sharper looking results.
Request bulletin 2799 on the reader response card.
Strep -tag Technology for Molecular Weight Determinations on
Blots Using Precision Plus Protein ™ Standards
Simplify your blotting protocols with Bio-Rad’s unstained Precision Plus Protein standards. These standards contain the unique Strep -tag affinity peptide on each protein, which allows detection of the standards directly on western blots. This tech note describes the mechanism of the Strep -tag detection system in detail. It contains images of the Precision Plus Protein unstained standards detected using Bio-Rad’s Opti-4CN ™ kit, Immun-Star ™ HRP kit, and
Immun-Blot ® AP kit, and describes the protocols used.
Request bulletin 2847 on the reader response card.
The Rotofor ® System Brochure
The Rotofor system fractionates complex protein samples by isoelectric focusing in free solution, often maintaining biological activity. This newly updated brochure describes how the Rotofor system works and how it can be used for preparative-scale fractionation.
Request bulletin 1903 on the reader response card.
Preparative Electrophoresis Product Folders
A variety of literature is available that describes applications using Bio-Rad’s preparative electrophoresis products, including the
Rotofor ® system (which fractionates protein samples by isoelectric focusing in free solution), the whole gel eluter (which elutes bands from preparative gels), and the Model 491 prep cell (which collects molecules as they migrate out of the bottom of a gel).
For the Rotofor technical folder, request bulletin1555A; for the Model 491 prep cell technical folder, request bulletin 1555B; for Bio-Rad’s whole gel eluter technical folder, request bulletin 1555C on the reader response card.
Beacon Designer is a trademark of PREMIER Biosoft Intl. Strep -tag is a trademark of
Institut für Bioanalytik GmbH. TaqMan is a trademark of Roche Molecular Systems.

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Precision Plus
Protein
Unstained
Standards
Precision Plus
Protein
All Blue
Standards
Precision Plus
Protein
Dual Color
Standards
50
37
25
20
15
10
250 kD
150
100
75
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For more information on Precision Plus Protein standards, contact your Bio-Rad representative or visit us at discover.bio-rad.com
Visit us on the Web at discover.bio-rad.com

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Visit us on the Web at discover.bio-rad.com
Call toll free at 1-800-4BIORAD (1-800-424-6723); outside the US, contact your local sales office.

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Visit us on the Web at discover.bio-rad.com
Call toll free at 1-800-4BIORAD (1-800-424-6723); outside the US, contact your local sales office.