www.bio-rad.com
Sample Quantitation
Cell Counting
24
Spectrophotometry
26
Fluorometry
30
Protein Assays
32
Cell Counting
www.bio-rad.com/cellcount/
Cell Counting
Researchers often need to count cells prior to cell culture or before starting processes and analytical techniques that demand
accurate and consistent numbers of input cells. These processes include transfection, cell proliferation or viability studies, and
quantitative PCR. Counting cells with a manual hemocytometer is a tedious task and results vary among researchers. To ensure
reliable results that are both accurate and reproducible, Bio-Rad offers an automated cell counter that simplifies cell counting.
New TC10™ Automated Cell Counter
The easy-to-use TC10 automated cell counter was developed
to count mammalian cells. The cell counter rapidly:
■■
■■
Provides a total cell count with or without staining
Assesses cell viability via the trypan blue dye exclusion
Accuracy is consistent and comparable to results obtained
with a manual hemocytometer when counting cells within
the 5 x 10 4 –1 x 107 cells/ml range and 6–50 μm cell
diameter, leading to highly reproducible cell counts. These
accurate and consistent counts lead to more reproducible
results with downstream processes and analysis.
For easy accessibility of data, results from 100 counts are
stored in the cell counter and can be exported via the USB
port and opened in an Excel spreadsheet. Images of the
cells can be viewed on the instrument; annotated JPEG
files can be exported via the USB port.
■■
■■
■■
■■
■■
■■
■■
One-step
counting — stand-alone instrument with
onboard software auto-counts immediately upon
slide insertion and auto-detects the presence of the
trypan blue dye (optional) in the sample with no user
input required
Trypan
blue dye optional — counts samples with or
without trypan blue dye
Small
sample volumes required — to conserve
precious cells, the counting slides require only ~10 μl of
sample suspended cells
TC10 Automated Cell Counter, Counting Slides,
Trypan Blue Dye
Dilution
calculator — calculates volume adjustments
required to achieve the cell concentration needed for
next passage/experiment
Fast
results — sophisticated software provides accurate
and reproducible cell counts within 30 sec
Auto-focus
— increases accuracy and saves time by
eliminating a manual focus step
Small
footprint — compact design takes up minimal
space (7 x 6") and fits inside a tissue culture hood
Use the TC10 automated cell counter to count samples with or
without trypan blue dye; cell viability is measured with the dye.
24 | Sample Quantitation
www.bio-rad.com
Cell Counting
www.bio-rad.com/cellcount/
One-Step Counting
Load
Insert
Results
>
Load the sample onto a slide.
>
Insert the slide into the TC10 counter;
counting begins automatically.
TC10™ Kits, Reagents, and Accessories
TC10 kits, reagents, and accessories are specially designed
and developed for use with the TC10 automated cell counter:
TC10 trypan blue dye — allows rapid staining
for accurate determination of cell viability. The dye has
been specially formulated for use with the TC10 counter
to provide maximum detection of viability
For researchers in regulated environments:
■■
■■
■■
TC10 counting slide — dual-chamber slide can provide
counts for two separate samples or dilutions; ensures
accurate and reproducible cell counts. Each chamber
requires only 10 µl, saving precious cells
■■
Thermal label printer — can be connected to the cell
counter for printing results onto labels that can be placed
into the laboratory notebook
■■
Obtain a total cell count (without trypan
blue dye) or total and live cell counts
(with trypan blue dye) in 30 seconds.
TC10 IQ/OQ kit — used to validate full functionality of the
TC10 cell counter and also serves as a positive control for
cell counting experiments; includes an IQ/OQ protocol and
an TC10 IQ/OQ slide
TC10 bar-coded counting slide — slide provides
traceability of counted samples by associating each
sample with a unique, 7 character bar code number
(read using the optional bar code reader). Bar codes
are stored in the software and associated with the
specific cell count and date of counting
TC10 bar code reader — offers traceability through
easy sample tracking
■■
Specifications
Counting time
Cell concentration range
Cell diameter range
30 seconds
5 x 104–1 x 107 cells/ml
6–50 μm
Dimensions (W x D x H)
19 x 16 x 25.4 cm (7.5 x 6 x 10")
Weight2.2 kg (without the external power
supply)
Ordering Information
Catalog #
Description
TC10 Automated Cell Counter
145-0001 TC10 Automated Cell Counter, 100–240 V, includes instrument, 30 TC10 counting slides with 2 chambers (60 count), 2 x 1.5 ml TC10 trypan blue dye
145-0002 TC10 Automated Cell Counter for Regulated Environments, 100–240 V, includes instrument, 30 TC10 bar-coded counting slides with 1 chamber (30 count), 1 x 1.5 ml TC10 trypan blue dye,
TC10 bar code reader, TC10 IQ/OQ kit, TC10 label printer, labels
Kits and Reagents
145-0003 TC10 Counting Kit, includes 30 TC10 counting slides with 2 chambers (60 count), 2 x 1.5 ml TC10 trypan blue dye
145-0004 TC10 Counting Kit for Regulated Environments, includes 30 TC10 bar-coded counting slides with 1 chamber (30 count), 1 x 1.5 ml TC10 trypan blue dye
145-0014 TC10 Installation Qualification and Operational Qualification Kit, includes IQ/OQ protocol, TC10 IQ/OQ slide
145-0013 TC10 Trypan Blue Dye, 0.4% in 0.81% sodium chloride and 0.06% potassium phosphate dibasic solution, sterile filtered, sufficient for 300 counts (10 µl/count), 2 x 1.5 ml
Accessories
145- 0011 145- 0012 145-0006 145-0005 145-0007 TC10 Counting Slides, dual chamber, 30 slides (60 samples)
TC10 Bar-Coded Counting Slides, single chamber, 30 slides (30 samples)
TC10 Bar Code Reader, 100–240 V TC10 Label Printer, 100–240 V Printer Labels, for TC10 label printer
www.bio-rad.com
Price, U.S. $
Inquire
Inquire
Inquire
Inquire
Inquire
Inquire
Inquire
Inquire
Inquire
Inquire
Inquire
Sample Quantitation | 25
Spectrophotometry
www.bio-rad.com/spectrophotometry/
Spectrophotometry
Bio-Rad offers a benchtop spectrophotometer with a large array of features for nucleic acid and protein quantitation,
along with a range of compatible cuvettes.
SmartSpec™ Plus Spectrophotometer
The SmartSpec Plus spectrophotometer has a more
complete range of features and functions than many
other benchtop spectrophotometers, offering affordable
performance, stability, and functionality.
The SmartSpec Plus is a UV/visible spectrophotometer
with a working wavelength range of 200–800 nm.
For routine work with nucleic acid and protein samples,
the SmartSpec Plus provides you with an instrument for:
Quantitation of DNA, RNA, oligonucleotides,
and proteins
■■
Monitoring cell culture growth
■■
Simple kinetics assays
■■
Wavelength scans with peak detection
A simple, menu-driven interface simplifies assays and
provides common sample computations at the touch of
a button. Conversion factors can be stored and modified.
The SmartSpec Plus instrument can automatically provide
results such as:
■■
A260/A280 ratio for nucleic acid purity
■■
Quantitation that takes dilution factors into account
■■
■■
Sample concentration in µg/ml (additionally in pmol/µl
for oligonucleotides)
Molar extinction coefficient and MW of oligonucleotides
At the end of an assay, a report may be printed that shows
the user, date, and results.
Nucleic Acid Quantitation
The SmartSpec Plus spectrophotometer offers a
complete solution for the quantitation of dsDNA,
ssDNA, or RNA by using either the preprogrammed
conversion factors or entering a value that is best
for the sample being assayed. The SmartSpec Plus
instrument will provide absorbance, concentration, and
purity values so you can proceed confidently with your
downstream experiments.
26 | Sample Quantitation
1
Absorbance
■■
0
225
Start wavelength = 225 nm
End wavelength = 350 nm
Max.: 261 nm = 0.724 OD
Min.: 349 nm = –0.014 OD
350
Wavelength, nm
Wavelength scan of a DNA sample from 225 to 350 nm.
The absorbance maximum is at 261 nm. The instrument can
be programmed to automatically subtract the background
absorbance.
Protein Quantitation
The SmartSpec Plus instrument has preprogrammed
methods for the quantitation of proteins by the Bradford,
Lowry, and BCA methods. Features built into each assay
method facilitate data collection and present a complete
analysis of assay results.
■■
Standards
can be analyzed in groups of up to
9 replicates
■■
Up
■■
Mean
■■
Printable report includes a standard curve with r2 value
to 10 standard curves can be stored under
user-assigned names
and standard deviation values are automatically
calculated for each replicate group
www.bio-rad.com
Spectrophotometry
www.bio-rad.com/spectrophotometry/
Other Benefits
Built-in thermal printer included
■■
■■
Xenon
flash lamp extends lamp life and reduces
maintenance costs
■■
User
■■
Compact, space-saving design
Cuvettes
Bio-Rad offers compatible quartz and UV-transparent
plastic cuvettes.
interface enables choice of 6 different languages:
English, French, German, Italian, Japanese, and Spanish
For more information, request bulletin 2826.
Spectrophotometer Cuvette Selection Guide
Min. Volume
Max. Volume
1,000 µl
500 µl
200 µl
80 µl
50 µl
3,500 µl 1,400 µl 700 µl
100 µl
1,500 µl
Cuvette Type
Pathlength
Catalog #
Standard cuvette, quartz
Semimicrovolume cuvette, quartz
Microvolume cuvette, quartz
Submicrovolume cuvette, quartz
trUView™ cuvette
10 mm
10 mm
10 mm
10 mm
10 mm
170-2502
170-2503
170-2504
170-2505
170-2510, 170-2511
Specifications
Light source
Xenon lamp
Absorption accuracy
±0.01 at 0.5 AU, ±0.02 at 1.0 AU
Detector
Photodiode array
Absorption repeatability
±0.005 at 0.5 AU, ±0.01 at 1.0 AU
Display
2 x 24 alphanumeric backlit LCD
Stray light
<0.2% at 230 nm using NaI standard
Printer
Built-in alphanumeric/graphic printer
Operating temperature range 15–35°C
Interface
RS-232 serial port
Beam height (z-dimension)
8.5 mm
Wavelength range
200–800 nm
Power requirement
90–260 VAC, 47–63 Hz
Wavelength accuracy
(at 18–27°C)
<±1.0 nm from 200 to 250,
300 to 800 nm; <±0.5 nm
from 250 to 300 nm
Regulatory
CE, TÜV
Dimensions (W x D x H)
33 x 26 x 14 cm
Spectral bandwidth
<5 nm
Weight
~3.8 kg
Ordering Information
Catalog #
Description
SmartSpec Plus Spectrophotometer
170-2525
SmartSpec Plus Spectrophotometer
Accessories
170-2502
170-2503
170-2504
170-2505
170-2510
170-2511
170-2506
170-2507
170-2512
Standard Cuvette, 1–3.5 ml, quartz
Semimicrovolume Cuvette, 0.5–1.4 ml, quartz
Microvolume Cuvette, 200–700 µl, quartz
Submicrovolume Cuvette, 80–100 µl, quartz
trUView Cuvettes, pack of 50, individually packaged, disposable DNase- and RNase-free cuvettes
trUView Cuvettes, pack of 100, individually packaged, disposable DNase- and RNase-free cuvettes
SmartSpec Printer Paper, 5 pack
Spring, cuvette holder
Height Adaptor, 15 mm, for trUView cuvettes
www.bio-rad.com
Price, U.S. $
5,471.00
158.00
235.00
243.00
489.00
58.00
120.00
39.00
25.00
35.00
Sample Quantitation | 27
Spectrophotometry
www.bio-rad.com/spectrophotometry/
trUView™ Cuvettes
These disposable cuvettes are suitable for most UV and
visible spectroscopic assays. Highly transparent trUView
cuvettes allow accurate and precise quantitation of
DNA, RNA, and protein. Individually packaged trUView
cuvettes are free of contaminants and are guaranteed
DNase, RNase, and pyrogen free. Features of trUView
cuvettes include:
■■
■■
Low volume requirement (≥50 µl) that conserves
limited samples
Individual packaging to prevent scratching and
contamination
trUView vs. Quartz Cuvettes
trUView cuvettes provide several advantages over
traditional quartz cuvettes for quantitative UV applications,
and have characteristics that can supplement or replace
the use of quartz cuvettes. trUView cuvettes are:
■■
■■
100
Up to 70% light transmission at 260 nm, ensuring
accurate nucleic acid quantitation
Ready to use without lengthy sterilization procedures
Disposable and less expensive to replace
80
% Transmission
■■
60
40
20
200
400
600
800
1,000
1,200
Wavelength, nm
Spectral scan of a trUView cuvette. Scan shows light transmission
from 220 to 1,100 nm. The high transmission at 260 nm allows accurate
quantitation of nucleic acids.
Specifications
Functional volume range
Pathlength
Functional wavelength range
Baseline absorbance
Variability
Outer dimensions (W x D x H)
50–1,500 µl
10 mm
220–1,100 nm
≤0.3 AU at 260 nm
≤15 mAU at 260 nm
12.5 x 12.5 x 45 mm
Ordering Information
Catalog #
Description
170-2510
170-2511
170-2512
trUView Cuvettes, pack of 50, individually packaged, disposable DNase- and RNase-free cuvettes
trUView Cuvettes, pack of 100, individually packaged, disposable DNase- and RNase-free cuvettes Height Adaptor, 15 mm, for trUView cuvettes
28 | Sample Quantitation
Price, U.S. $
58.00
120.00
35.00
www.bio-rad.com
Spectrophotometry
www.bio-rad.com/spectrophotometry/
Standard and Semimicrovolume Cuvettes
Standard 3.5 ml cuvettes are ideal for use with protein
assays (pages 32–37). They adsorb less Coomassie Blue
dye than glass or quartz cuvettes. Assays can be mixed
directly in the cuvette. The volume of reagents can be
reduced, yielding more assays per kit. Semimicrovolume
1.5 ml cuvettes are ideal for precise quantitation of small
volume samples. Both standard and semimicrovolume
cuvettes are sized to fit most spectrophotometers. The
cuvettes offer smooth optical surfaces for consistent and
accurate readings.
100
80
% Transmission
Bio-Rad also offers other cuvettes for specialized
applications, including quartz and disposable cuvettes for
SmartSpec™ spectrophotometers, disposable cuvettes
designed for the VersaFluor™ fluorometer, and disposable
trUView™ cuvettes for precise quantitation of small samples
of DNA, RNA, and proteins.
60
40
20
0
300
400
500
600
700
800
Wavelength, nm
Spectral scan of Bio-Rad’s standard cuvette.
Specifications
Energy levels
80% T at 800 nm
55% T at 340 nm
Material
Premium-grade virgin polystyrene
Dimensions
12.5 mm2 x 45 mm high
Transmission matching
±1%
Capacity
Pathlength matching
±0.5%
Pathlength
1 cm
Standard
Semimicro
3.5 ml
1.5 ml
Ordering Information
Catalog #
Description
Disposable Polystyrene Cuvettes
223-9950
Standard Disposable Polystyrene Cuvettes, 3.5 ml, 100
223-9955
Semimicrovolume Disposable Polystyrene Cuvettes, 1.5 ml, 100
Price, U.S. $
12.00
12.00
Quartz Cuvettes
170-2502
Standard Cuvette, 1–3.5 ml, quartz, for use with the SmartSpec Plus spectrophotometer
170-2503
Semimicrovolume Cuvette, 0.5–1.4 ml, quartz, for use with the SmartSpec Plus spectrophotometer
170-2504
Microvolume Cuvette, 200–700 µl, quartz, for use with the SmartSpec Plus spectrophotometer
170-2505
Submicrovolume Cuvette, 80–100 µl, quartz, for use with the SmartSpec Plus spectrophotometer 158.00
235.00
243.00
489.00
trUView Cuvettes
170-2510
trUView Cuvettes, pack of 50
170-2511
trUView Cuvettes, pack of 100
58.00
120.00
VersaFluor Disposable Cuvettes
170-2415
Standard Cuvettes, 12.5 x 12.5 mm (outside dimensions), 4-sided, optically clear polystyrene, 3.5 ml, 100
170-2416
Microcuvettes, 3.0 x 3.0 mm (outside dimensions), 4-sided optically clear polystyrene, 150–350 µl, 100
25.00
35.00
Accessories
170-2406
170-2407
170-2512
63.00
49.00
35.00
Standard Cuvette Holder
Microcuvette Holder
Height Adaptor, 15 mm, for trUView cuvettes Inquire about tiered pricing for bulk purchases.
www.bio-rad.com
Sample Quantitation | 29
Fluorometry
www.bio-rad.com/fluorometry/
Fluorometry
Bio-Rad offers a benchtop fluorometer that is compatible with a number of different filters, along with several
fluorescence assay reagents for DNA quantitation and reporter gene assays.
VersaFluor™ Fluorometer
The VersaFluor fluorometer is a flexible and affordable
benchtop fluorometer, featuring user-selectable filters
for precise sample excitation and emission analysis.
The VersaFluor fluorometer can excite and detect a broad
spectrum of fluorescent dyes anywhere in the range of
350–900 nm. Range and gain settings are adjustable
to maximize sensitivity. The instrument is compact and
easy to operate using menu-driven software. Data can be
exported through the RS-232 serial port to the optional
thermal printer or to a PC.
High Sensitivity and Selectivity for Any
Fluorescence Assay
The VersaFluor fluorometer quantitates DNA accurately at
concentrations as low as 10 ng/ml using Hoechst dye in a
standard 2 ml assay. Hoechst dye selectively binds dsDNA
in the presence of RNA, proteins, or nucleotides, offering
an affordable solution to the sensitivity and selectivity
required for applications such as real-time PCR.
The VersaFluor fluorometer allows:
■■
■■
■■
Accurate quantitation of DNA, RNA, oligonucleotides,
and proteins
Performance of reporter gene assays (b-galactosidase,
b-glucuronidase, or GFP activity)
Detection of apoptosis
■■
■■
Use of either disposable plastic or quartz cuvettes for
sample analysis
Use of standard cuvettes (1.0–3.5 ml) or microcuvettes
(150–300 µl)
Multiple Filters Available
User-selectable filters support a multitude of applications
with a broad range of excitation and emission wavelengths.
Custom filter sets are also available from your local Bio-Rad
sales representative. The following table lists commonly
used fluorescence assays and recommended excitation
and emission filters.
For more information, request bulletin 2234.
Fluorescence Assay Filter Selection Guide
Application
Excitation Filter
Catalog #
Emission Filter
Catalog #
EX 360/40 (340–380 nm)
EX 360/40 (340–380 nm)
EX 360/40 (340–380 nm)
EX 480/20 (470–490 nm)
EX 490/10 (485–495 nm)
EX 480/20 (470–490 nm)
EX 480/20 (470–490 nm)
EX 490/10 (485–495 nm)
EX 510/10 (505–515 nm)
EX 390/22 (379–401 nm)
EX 490/10 (485–495 nm)
EX 380/30 (365–395 nm)
EX 390/22 (379–401 nm)
170-2420
170-2420
170-2420
170-2427
170-2422
170-2427
170-2427
170-2422
170-2423
170-2443
170-2422
170-2444
170-2443
EM 460/10 (455–465 nm)
EM 460/10 (455–465 nm)
EM 460/10 (455–465 nm)
EM 520/10 (515–525 nm)
EM 520/10 (515–525 nm)
EM 520/10 (515–525 nm)
EM 590/10 (585–595 nm)
EM 520/10 (515–525 nm)
EM 620/10 (615–625 nm)
EM 510/10 (505–515 nm)
EM 510/10 (505–515 nm)
EM 440/10 (435–445 nm)
EM 510/10 (505–515 nm)
170-2421
170-2421
170-2421
170-2424
170-2424
170-2424
170-2425
170-2424
170-2426
170-2445
170-2445
170-2446
170-2445
DNA quantitation (Hoechst 33258)
b-galactosidase (MUG) b-glucuronidase (MUGluc) DNA quantitation (PicoGreen) RNA quantitation (RiboGreen)
Oligonucleotide/protein quantitation (OliGreen)
Protein quantitation (NanoOrange)
Fluorescein
Ethidium bromide
wtGFP (wild-type green fluorescent protein) EGFP (red-shifted green fluorescent protein)
EBFP (blue-emitting green fluorescent protein)
GFPuv (UV-excited green fluorescent protein)
30 | Sample Quantitation
www.bio-rad.com
Fluorometry
www.bio-rad.com/fluorometry/
Ordering Information
Catalog #
Description
Price, U.S. $
VersaFluor Fluorometer
170-2402VersaFluor Fluorometer, 100/120/220 V, includes 1 standard cuvette holder, 100 standard cuvettes, choice of 1 excitation and 1 emission filter
4,329.00
Filter Sets, Excitation and Emission*
170-2435Filter Set (EX 360/40, EM 460/10), for DNA quantitation (Hoechst 33258), b-galactosidase (MUG), and
b-glucuronidase (MUGluc) assays
170-2436
Filter Set (EX 490/10, EM 520/10), for fluorescein-based assays
170-2437
Filter Set (EX 360/40, EM 520/10)
170-2447
Filter Set (EX 390/2, EM 510/10), for wild-type GFP and UV-excited GFP
493.00
493.00
511.00
Individual Excitation Filters
170-2420
EX 360/40 (340–380 nm)
170-2427
EX 480/20 (470–490 nm)
170-2422
EX 490/10 (485–495 nm)
170-2423
EX 510/10 (505–515 nm)
170-2443
EX 390/22 (379–401 nm)
170-2444
EX 380/30 (365–395 nm)
266.00
266.00
266.00
266.00
266.00
266.00
Individual Emission Filters
170-2421
EM 460/10 (455–465 nm)
170-2424
EM 520/10 (515–525 nm)
170-2425
EM 590/10 (585–595 nm)
170-2426
EM 620/10 (615–625 nm)
170-2445
EM 510/10 (505–515 nm)
170-2446
EM 440/10 (435–445 nm)
266.00
266.00
266.00
266.00
266.00
266.00
VersaFluor Disposable Cuvettes
170-2415
Standard Cuvettes, 4-sided optically clear polystyrene, 3.5 ml, 12.5 x 12.5 mm OD, 100
170-2416
Microcuvettes, 4-sided optically clear polystyrene, 150–350 µl, 3.0 x 3.0 mm OD, 100
Accessories
170-2406
170-2407
170-2430
170-2450
170-2412
Standard Cuvette Holder, for a single 4-sided 3.5 ml standard cuvette
Microcuvette Holder, for a single 4-sided microcuvette
Quartz Halogen Replacement Lamp
Optical Filter Storage Case, holds 12 filters
Thermal Printer Paper, 10 rolls
493.00
25.00
35.00
63.00
49.00
40.00
Inquire
128.00
* Contact your local Bio-Rad sales representative for availability of custom filters.
Fluorescence Assay Kits
DNA Quantitation Kit
Quickly and accurately quantitate double-stranded DNA
in solution — even in the presence of RNA or protein —
with Bio-Rad’s fluorescence DNA quantitation kit. DNA
is stained with a bisbenzimide dye (Hoechst 33258) and
excited at 346 nm to emit light at 460 nm, which is easily
detected by most fluorometers. The kit quantitates
dsDNA in the range of 10 ng/ml to 5 µg/ml.
FluorAce b-Glucuronidase Reporter Assay Kit
Detect b-glucuronidase activity quickly and precisely
with the FluorAce b-glucuronidase reporter assay kit.
The substrate 4-methylumbelliferyl b-D-glucuronide
(MUGluc) is cleaved by b-glucuronidase, producing a
fluorogenic compound that is easily detected with most
fluorometers.
FluorAce™ b-Galactosidase Reporter Assay Kit
Detect b-galactosidase activity easily and accurately
with the FluorAce b-galactosidase reporter assay kit.
Simply add the fluorescent substrate 4-methylumbelliferyl
b-D-galactopyranoside (MUG) to cell culture medium.
Cellular MUG is cleaved by b-galactosidase, producing
4-methylumbelliferone (4MU), which is easily detected by
most fluorometers.
www.bio-rad.com
Sample Quantitation | 31
Protein Assays
www.bio-rad.com/proteinassays/
Ordering Information
Catalog #
Description
Price, U.S. $
170-2480DNA Quantitation Kit, includes Hoechst 33258, calf thymus DNA standard, assay buffer; sufficient for 750 standard 2 ml reactions
170-3150FluorAce b-Galactosidase Reporter Assay Kit, includes reaction buffer, MUG substrate, stop buffer, 4MU control, b-galactosidase; sufficient for 200 standard 1.5 ml reactions
170-3151FluorAce b-Glucuronidase Reporter Assay Kit, includes reaction buffer, MUGIuc substrate, stop buffer, 4MU control, b-glucuronidase; sufficient for 200 standard 1.5 ml reactions
101.00
153.00
199.00
Protein Assays
See Also
SmartSpec Plus
spectro­photometer:
pages 26–27.
Microplate readers:
pages 366–368.
Cuvettes:
pages 28–29.
Determining the concentration of protein samples is critical for many experiments, and most protein samples can be
quantitated by a colorimetric assay. In a typical protein assay, a chemical reagent is added to a protein sample solution,
producing a color change that is measured with a spectrophotometer or microplate reader and compared to a standard
curve of known protein concentrations. Bio-Rad offers four protein assays, each with a unique set of advantages. All assays
can be easily automated for large numbers of samples.
Bio-Rad offers four protein assays:
■■
■■
■■
■■
Start™
Quick
Bradford protein assay — the single-step
method for determining protein concentration in solution
Bio-Rad protein assay — for quantitating most proteins
and polypeptides with MWs >3,000
C™ protein assay — for use following detergent
D
solubilization
RC DC™ protein assay — for complex sample solutions
containing reducing agents and detergents
Choosing a Protein Assay
When selecting a protein assay method, the chemical
components contained in the buffer and the amount of protein
available for assay are important to consider. The following
table summarizes characteristics of each protein assay.
Specific chemical compatibilities for each assay are listed with
each product. All assays are easy to use, require little reagent
preparation, and give accurate and reproducible results.
Choosing a Protein Standard
In any protein assay, the best standard to use is a purified
preparation of the protein being assayed. In the absence
of such an absolute reference protein, the best relative
standard to use is one that gives a color yield similar to
that of the protein being assayed. If only relative protein
concentrations are needed, any purified protein can be
selected as a reference standard.
Bio-Rad offers two protein standards, bovine serum albumin
and bovine g-globulin, in lyophilized and prediluted formats.
For more information, request bulletin 1069.
Protein Assay Selection Guide*
Quick Start Bradford
Bio-Rad
DC
RC DC
Method adapted from
Bradford (1976)
Bradford (1976)
Lowry et al. (1951)
Lowry et al. (1951)
Standard-concentration assay
Sample volume
Linear range
100 µl
0.125–1.5 mg/ml
100 µl
0.125–1.5 mg/ml
100 µl
0.125–1.5 mg/ml
100 µl
0.2–1.5 mg/ml
Low-concentration assay
Sample volume
Linear range
1 ml
1.25–25 µg/ml
800 µl
1.25–25 µg/ml
200 µl
5–250 µg/ml
200 µl
5–250 µg/ml
Microplate assay sample volume
5 µl
10 µl
5 µl
**
Minimum incubation time
5 min
5 min
15 min
15 min
Assay wavelength
595 nm
595 nm
650–750 nm
650–750 nm
* Standard-concentration assay is designed for smaller sample volumes of higher protein concentration, while the low-concentration assay is
designed for larger sample volumes of lower protein concentration.
** To adapt the RC DC assay to a microplate format, follow the micro test tube (microfuge tube) assay protocol in the RC DC protein assay instruction
manual up to the centrifugation step where the supernatant is discarded. The pellet can then be transferred to the microplate, and the microplate
assay protocol in the DC protein assay instruction manual can be followed.
32 | Sample Quantitation
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Protein Assays
www.bio-rad.com/proteinassays/
Quick Start™ Bradford Protein Assay
The Quick Start Bradford protein assay is a streamlined,
accurate procedure for determining the concentration
of protein in solution. Ready-to-use dye reagent and
prediluted protein standards allow one-step determination
of protein concentration.
Quick Start Bradford kits offer either bovine serum albumin
or bovine g-globulin standard sets. Each kit contains two
aliquots of seven concentrations (0.125, 0.25, 0.5, 0.75, 1.0,
1.5, 2.0 mg/ml) conveniently packaged in screwcap vials,
eliminating ampoules and ensuring protein stability for one
year when stored at 4°C. Standards are also available in
five 2 mg/ml aliquots for creating your own dilutions.
The 1x dye reagent can be used for performing 1 ml and
5 ml standard assays, microplate assays, or microassays.
For more information, request bulletin 1069.
Compatible Reagents*
Acetone, 10%
Ethanol, 10%
Octyl b-thioglucopyranoside, 1%
Sucrose, 10%
Acetonitrile, 10%
Glucose, 20%
PBS
Ammonium sulfate, 1 M
Glycerol, 5%
Phenol red, 0.5 mg/ml
TBS, 0.5x (12.5 mM Tris, 75 mM
NaCl, pH 7.6)
Ampholytes, pH 3–10, 0.5%
Glycine, 0.1 M
PIPES, 0.2 M
TCEP, 20 mM
ASB-14, 0.025%
Guanidine-HCl, 2 M
PMSF, 2 mM
Thiourea, 1 M
Ascorbic acid, 50 mM
Hank’s salt solution
Potassium chloride, 2 M
Tributylphosphine (TBP), 5 mM
Bis-Tris, pH 6.5, 0.2 M
HCl, 0.1 M
Potassium phosphate, 0.5 M
Tricine, pH 8, 50 mM
Calcium chloride, 40 mM
HEPES, 0.1 M
SB 3-10, 0.1%
Triethanolamine, pH 7.8, 50 mM
CHAPS, 10%
Imidazole, 0.2 M
SDS, 0.025%
Tris, 1 M
CHAPSO, 10%
Magnesium chloride, 1 M
Sodium acetate, pH 4.8, 0.2 M
Deoxycholic acid, 0.2%
b-mercaptoethanol, 1 M
Sodium azide, 0.5%
Tris-glycine (25 mM Tris,
192 mM glycine)
Dithioerythritol (DTE), 10 mM
MES, 0.1 M
Sodium bicarbonate, 0.2 M
Dithiothreitol (DTT), 10 mM
Methanol, 10%
Sodium carbonate, 0.1 M
Tris-glycine-SDS, 0.25x
(6.25 mM Tris, 48 mM glycine,
0.025% SDS)
DMSO, 5%
Modified Dulbecco’s PBS
Sodium chloride, 2.5 M
Triton X-100, 0.05%
Eagle’s MEM
MOPS, 0.1 M
Tween 20, 0.01%
Earle’s salt solution
NAD, 2 mM
Sodium citrate, pH 4.8 or 6.4,
0.2 M
EDTA, 0.2 M
Nonidet P-40 (NP-40), 0.25%
Sodium hydroxide, 0.1 M
EGTA, 0.2 M
Octyl-b-glucoside, 0.5%
Sodium phosphate, 0.5 M
Urea, 4 M
* Concentrations compatible with the microassays are 1/25 of the values listed.
Ordering Information
Catalog #
Description
Quick Start Bradford Protein Assay
500-0201Quick Start Bradford Protein Assay Kit 1, includes 1x dye reagent (1 L), bovine serum albumin standard (5 x 2 mg/ml); sufficient for 200 standard assays or 4,000 microplate assays
500-0202Quick Start Bradford Protein Assay Kit 2, includes 1x dye reagent (1 L), bovine serum albumin standard set (2 sets of 7 concentration standards, 0.125–2.0 mg/ml, 2 ml)
500-0203
Quick Start Bradford Protein Assay Kit 3, includes 1x dye reagent (1 L), bovine g-globulin standard (5 x 2 mg/ml)
500-0204
Quick Start Bradford Protein Assay Kit 4, includes 1x dye reagent (1 L), bovine g-globulin standard set (2 sets of 7 concentration standards, 0.125–2.0 mg/ml, 2 ml)
Price, U.S. $
115.00
143.00
119.00
147.00
continues
www.bio-rad.com
Sample Quantitation | 33
Protein Assays
www.bio-rad.com/proteinassays/
Ordering Information
Catalog #
Description
Reagents
500-0205
Price, U.S. $
Quick Start Bradford 1x Dye Reagent, 1 L
Accessories
500-0206
500-0207
500-0208
500-0209
223-9950
223-9955
Quick Start Bovine Serum Albumin Standard, 5 x 2 ml vials of 2 mg/ml
Quick Start Bovine Serum Albumin Standard Set, 2 sets of 7 concentration standards, 0.125–2.0 mg/ml
Quick Start Bovine g-Globulin Standard, 5 x 2 ml vials of 2 mg/ml
Quick Start Bovine g-Globulin Standard Set, 2 sets of 7 concentration standards, 0.125–2.0 mg/ml
Standard Disposable Polystyrene Cuvettes, 3.5 ml, 100
Semimicrovolume Disposable Polystyrene Cuvettes, 1.5 ml, 100
97.00
64.00
107.00
65.00
109.00
12.00
12.00
Bio-Rad Protein Assay
The Bio-Rad protein assay is a simple colorimetric assay
for determining total protein concentration. It is easy to
adapt the assay from the standard-concentration range
to a low-concentration microassay or for use in 96-well
microplates for rapid determinations.
The Bio-Rad protein assay is based on the Bradford dyebinding procedure (Bradford 1976), which measures the
color change of Coomassie Brilliant Blue G-250 dye when
it binds to protein (primarily to basic and aromatic amino
acid residues). The assay quantitates most proteins and
polypeptides with molecular weights >3,000–5,000. Many
detergents and basic buffers interfere with the assay; refer
to the list of chemical reagents (below) that do not directly
affect the development of dye color. Since not every proteinreagent combination has been tested, some reagents listed
may interfere with the assay of certain proteins.
For more information, request bulletins 1069 and 1123.
Compatible Reagents*
Acetate, 0.6 M
Acetone
Acid pH
Adenosine, 1 mM
Amino acids
Ammonium sulfate, 1 M
Ampholytes, pH 3–10, 0.5%
ATP, 1 mM
Barbital
BES, 2.5 M
Boric acid
Cacodylate-Tris, 0.1 M
CDTA, 50 mM
Citrate, 50 mM
Deoxycholate, 0.1%
Dithiothreitol (DTT), 1 M
DNA, 1 mg/ml
Eagle’s MEM
Earle’s salt solution
EDTA, 0.1 M
EGTA, 50 mM
Ethanol
Formic acid, 1 M
Fructose
Glucose
Glutathione
Glycerol, 99%
Glycine, 0.1 M
Guanidine-HCl
Hank’s salt solution
HEPES, 0.1 M
Magnesium chloride, 1 M
Malic acid, 0.2 M
b-mercaptoethanol, 1 M
MES, 0.7 M
Methanol
MOPS, 0.2 M
NAD, 1 mM
NaSCN, 3 M
Peptones
Phenol, 5%
Phosphate, 1 M
PIPES, 0.5 M
Polyadenylic acid, 1 mM
Polypeptides (MW <3,000)
Potassium chloride, 1 M
Pyrophosphate, 0.2 M
rRNA, 0.25 mg/ml
tRNA, 0.4 mg/ml
Total RNA, 0.3 mg/ml
SDS, 0.1%
Sodium chloride, 5 M
Sodium phosphate
Streptomycin sulfate, 20%
Thymidine, 1 mM
Tricine
Tris, 2 M
Triton X-100, 0.1%
Tyrosine, 1 mM
Urea, 6 M
Vitamins
* The concentration limits are for the sample before dilution with the dye reagent using the standard procedure. Where concentrations are not given, assume standard concentrations in culture media. Interference in other formats is proportional to the sample concentration. Concentrations compatible with the low-concentration assay procedure (1.25–25 µg/ml protein) are 1/40 of the values listed.
34 | Sample Quantitation
www.bio-rad.com
Protein Assays
www.bio-rad.com/proteinassays/
Bio-Rad Protein Assay Formats
Test Tube
Microplate
High-concentration assay
0.2–1.4 mg/ml protein
100 µl sample
5 ml diluted reagent
0.05–0.5 mg/ml protein
10 µl sample
200 µl diluted reagent
Low-concentration assay
1.25–25 µg/ml protein
800 µl sample
200 µl reagent concentrate
8–80 µg/ml protein
160 µl sample
40 µl reagent concentrate
Ordering Information
Catalog #
Description
Price, U.S. $
Bio-Rad Protein Assay
500-0001
Bio-Rad Protein Assay Kit I, includes 450 ml dye reagent concentrate, bovine g-globulin standard; sufficient for 440 standard assays or 2,200 microplate assays
500-0002
Bio-Rad Protein Assay Kit II, includes 450 ml dye reagent concentrate, bovine serum albumin standard; sufficient for 440 standard assays or 2,200 microplate assays
113.00
113.00
Reagents
500-0006
Bio-Rad Protein Assay Dye Reagent Concentrate, 450 ml
99.00
Accessories
500-0005
500-0007
223-9950
223-9955
Protein Standard I, bovine g-globulin, reconstituted volume 20 ml
Protein Standard II, bovine serum albumin, reconstituted volume 20 ml
Standard Disposable Polystyrene Cuvettes, 3.5 ml, 100
Semimicrovolume Disposable Polystyrene Cuvettes, 1.5 ml, 100
59.00
59.00
12.00
12.00
DC™ Protein Assay
The DC (detergent compatible) protein assay is a colorimetric
assay for protein determination following detergent
solubilization. The reaction is similar to the well-documented
Lowry assay (Lowry et al. 1951) but has been modified to
save time. The DC protein assay requires only a single
15 minute incubation, and the absorbance readings are
stable for at least 2 hours.
For more information, request bulletins 1069 and 1909.
Compatible Reagents*
Brij 35, 1%
C12E8,** 0.2%
Calcium chloride, 50 mM
CHAPS, 1%
CHAPSO, 1%
Dithiothreitol (DTT), 1 mM
EDTA, <1 mM
Guanidine-HCl, 0.4 M
HCl, 0.5 M
(NH4)2SO4, 0.5 M
Nonidet P-40 (NP-40), 2%
Octyl-b-glucoside, 1%
SDS, 10%
Sodium azide, 0.05%
Sodium hydroxide, 0.5 M
Thesit, 1%
Tris, pH 8, 0.1 M
Triton X-100, 1%
Tween 20, 1%
Urea, 4 M
* Using standard procedure.
** Octaethyleneglycol dodecyl ether.
DC Protein Assay Formats
Test Tube
Microplate
High-concentration assay
0.2–1.5 mg/ml protein
100 µl sample
500 µl reagent A
4.0 ml reagent B
0.2–1.5 mg/ml protein
5 µl sample
25 µl reagent A
200 µl reagent B
Low-concentration assay
5–250 µg/ml protein
200 µl sample
100 µl reagent A
800 µl reagent B
5–250 µg/ml protein
20 µl sample
10 µl reagent A
80 µl reagent B
www.bio-rad.com
Sample Quantitation | 35
Protein Assays
www.bio-rad.com/proteinassays/
Ordering Information
Catalog #
Description
Price, U.S. $
DC Protein Assay
500-0111
DC Protein Assay Kit I, includes 250 ml alkaline copper tartrate solution, 2 L dilute Folin reagent, 5 ml surfactant solution, bovine g-globulin standard; sufficient for 450 standard assays
500-0112
DC Protein Assay Kit II, includes 250 ml alkaline copper tartrate solution, 2 L dilute Folin reagent, 5 ml surfactant solution, bovine serum albumin standard; sufficient for 450 standard assays
146.00
146.00
Reagents
500-0113
Protein Assay Reagent A, 250 ml alkaline copper tartrate solution
500-0114
Protein Assay Reagent B, 1 L dilute Folin reagent
500-0115
Protein Assay Reagent S, 5 ml surfactant solution
500-0116DC Protein Assay Reagents Package, includes 250 ml alkaline copper tartrate solution, 2 L dilute Folin reagent, 5 ml surfactant solution; sufficient for 450 standard assays
Accessories
500-0005
500-0007
223-9950
223-9955
See Also
Protein sample
preparation:
pages 11–22.
Cuvettes:
pages 28–29.
46.00
46.00
31.00
141.00
Protein Standard I, bovine g-globulin, lyophilized (reconstituted volume 20 ml)
Protein Standard II, bovine serum albumin, lyophilized (reconstituted volume 20 ml)
Standard Disposable Polystyrene Cuvettes, 3.5 ml, 100
Semimicrovolume Disposable Polystyrene Cuvettes, 1.5 ml, 100
59.00
59.00
12.00
12.00
RC DC™ Protein Assay
The RC DC (reducing agent and detergent compatible)
protein assay is a colorimetric assay for protein
determination in the presence of reducing agents and
detergents. The RC DC protein assay, based on the
Lowry protocol (Lowry et al. 1951), includes the features
of the original DC™ protein assay. Its compatibility with
a broader range of reagents allows simplified protein
quantitation directly in complex sample solutions.
For more information, request bulletins 1069 and 2610.
Compatible Reagents*
The RC DC protein assay is compatible with the following reagents and buffers in addition to those compatible with the DC protein assay:
b-mercaptoethanol, 10%
ReadyPrep™ sequential extraction
reagents 2 and 3 (see pages 15–16)
Sodium hydroxide, 2.5 M
TBP, 2 mM
Tris, pH 8.4, 0.5 M
Triton X-100, 2%
Tween 20, 2%
Test Tube
Micro Test Tube
High-concentration assay
0.2–1.5 mg/ml protein
100 µl sample
500 µl reagent I
500 µl reagent II
500 µl reagent A
4.0 ml reagent B
0.2–1.5 mg/ml protein
25 µl sample
125 µl reagent I
125 µl reagent II
125 µl reagent A
1.0 ml reagent B
CHAPS, 2%
Dithiothreitol (DTT), 350 mM
EDTA, 0.1 M
Imidazole, 0.5 M
Laemmli buffer with 5% b-mercaptoethanol
* Using standard procedure.
RC DC Protein Assay Formats
36 | Sample Quantitation
www.bio-rad.com
Protein Assays
www.bio-rad.com/proteinassays/
Ordering Information
Catalog #
Description
RC DC Protein Assay
500-0121
RC DC Protein Assay Kit I, includes RC reagents package, DC protein assay reagents package, bovine g-globulin standard; sufficient for 450 standard assays
500-0122
RC DC Protein Assay Kit II, includes RC reagents package, DC protein assay reagents package, bovine serum albumin standard; sufficient for 450 standard assays
Reagents
500-0113
500-0114
500-0115
500-0116
500-0120
500-0119
500-0117
500-0118
Protein Assay Reagent A, 250 ml alkaline copper tartrate solution
Protein Assay Reagent B, 1 L dilute Folin reagent
Protein Assay Reagent S, 5 ml surfactant solution
DC Protein Assay Reagents Package, includes 250 ml alkaline copper tartrate solution, 2 L dilute Folin reagent, 5 ml surfactant solution; sufficient for 450 standard assays
RC DC Protein Assay Reagents Package, includes RC reagents package and DC protein assay reagents package; sufficient for 450 standard assays
RC Reagents Package, includes RC reagent I (250 ml) and RC reagent II (250 ml); sufficient for 500 standard assays
RC Reagent I, 250 ml
RC Reagent II, 250 ml
Accessories
500-0005
500-0007
223-9950
223-9955
Protein Standard I, bovine g-globulin, lyophilized (reconstituted volume 20 ml)
Protein Standard II, bovine serum albumin, lyophilized (reconstituted volume 20 ml)
Standard Disposable Polystyrene Cuvettes, 3.5 ml, 100
Semimicrovolume Disposable Polystyrene Cuvettes, 1.5 ml, 100
www.bio-rad.com
Price, U.S. $
227.00
227.00
46.00
46.00
31.00
141.00
223.00
91.00
50.00
50.00
59.00
59.00
12.00
12.00
Sample Quantitation | 37
38 |
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