Issue 1
April 2015
I Page10 FectoPRO™: Achieve Amazing Protein Yields I Page 20 Rapid, High-Performance, and
Cost-Effective Plant DNA Extractions I Page 26 qTOWER 2.0/2.2: Set New Standards in
Real-Time Quantitative PCR I Page 32 Even the Smallest Thing Can Have a Big Impact
CONTENTS
CeLl Biology
Fetal Bovine Serum: What You Should Ask Your Supplier and Why . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Corning® Cytokines, Growth Factors, and Other Media Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
siRNA Dependent Gene Silencing in HeLa Cells Cultivated on Various Cell Culture Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
FectoPRO™: Achieve Amazing Protein Yields in CHO and HEK-293 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Understanding the Molecular Basis of Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Intrawell Cell Distribution in Nunc® Microwell Edge Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Genomics
Unique Enzymes from the Arctic Alleviate the Need to Purify Samples for Several Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
qScript™ XLT SuperMix Superior cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Rapid, High-Performance, and Cost-Effective Plant DNA Extractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Robust and Highly-Specific Multiplex PCR Using Q5® High-Fidelity DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
FlashGel™ System for DNA Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
qTOWER 2.0/2.2: Set New Standards in Real-time Quantitative PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Using the DuPont™ BAX® System to Detect Salmonella, E. coli O157:H7 and non-O157 STEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
OPTIMIZER PCR Workstation™ Improves Accuracy of Sensitive PCR Amplification Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Proteomics
Even the Smallest Thing Can Have a Big Impact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Sensitive Detection of Horseradish Peroxidase (HRP) for Western Blotting Detection of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Azure Biosystems Presents the Only Imaging System for all Western Blot Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Coomassie Protein Staining with Thermo Scientific™ Pierce™ Power Stainer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Multiplex Fluorescent Western Blot Detection Using BioSpectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Inside the Biotix Robotic Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
2
VWR International I VWRbioMarke Issue 1 I April 2015
CeLL Biology
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Fetal Bovine Serum:
What You Should Ask
Your Supplier and Why
Devin Davis and Sherrie Drake Hirschi
BioProcess J, 2014; 13(1): 19-21. http://dx.doi.org/10.12665/J131
In today’s volatile sera market, it is critical
that sera users worldwide thoroughly
review their supply relationships and update
sourcing and risk mitigation strategies.
BioProcessing Journal’s recent article by
Siegel and Foster highlighted the impact
of selecting the appropriate country of
origin as one criterion for purchasing
decisions.1 Many more vital selection
criteria exist to ensure a sera supplier
provides long-term assurance of supply
and integrity of supply. This article
identifies critical questions sera
users should ask their suppliers
and explains why they should
ask them.
Changing Market Dynamics
Necessitate Review
The sera industry has experienced
substantial changes in the last year.
Mergers and divestitures have
introduced instability to, and turnover
of, once stable supply relationships.
The first mass recall of fetal bovine
serum (FBS) in history for adulterated
product impacted sera suppliers
and users.2 The World
Organisation for Animal
Health (OIE) announced the
risk status of bovine
spongiform encephalopathy
(BSE) upgrade in the United
States to “negligible risk,”
establishing US-origin FBS as
equal in safety to that of
Australia and New Zealand.3
In addition, new industry
standards for quality and
traceability have been
established to inform, protect,
and support sera users in
selecting a sera supplier.4
These historic changes — combined with
historically low cattle inventory levels5 —
demonstrate that the traditional paradigm for
sourcing sera is no longer valid. Sera users can
use the following discussion points to kickstart a conversation with existing and
potential sera suppliers to ensure their
research or production requirements are
uninterrupted by these dynamics.
Discussion Point #1
Assurance of Supply
Assurance of supply resides at the source—the
abattoirs where the raw materials for sera
products are procured. Sera suppliers attempt
to provide sourcing stability to their customers
by establishing strong relationships at the
source. Many of these relationships have
shifted due to the merger and divestiture
activity of the past year.
Similarly, a sera supplier’s relationships at
the source have a dramatic impact on the
quality of products they supply. Product
quality indicators—such as endotoxin and
hemoglobin levels—are driven by the care
and attention given to the raw materials
when initially collected and processed.
Not all abattoirs, collection techniques,
and raw material processing steps are
created equal.
Reputable sera suppliers take the time to
educate their customers about their supply
relationships, collection and processing
steps, and any changes that have occurred as a
result of the market dynamics previously
mentioned. Sera users should thoroughly
investigate the ability of a supplier to provide
long-term assurance of supply. This
investigation may validate current sourcing
practices or uncover sourcing risks that were
not previously known.
April 2015 I VWRbioMarke Issue 1 I VWR International
3
Assurance of Supply
What Questions to Ask
Why is it Important
Describe your supply relationships at the source.
Allows the supplier to articulate their story.
Where do you collect? Which beef packers do you work with? Why do you work with them?
Quantifies the scope of their supply chain and why they are organized that way.
Are you single-sourced or multi-sourced?
Quantifies the risk of interruptions given reduced product availability. Multi-sourced is more secure.
Do you do the work yourself or involve a partner? Why do you do it this way?
Pros and cons exist for both vertically integrated or outsourced supply chains. Find out why the
supplier prefers their approach and which certified partners they work with.
What kind of agreements are in place?
Understand what type and length of contracts are in place to reduce the risk of supply
interruptions.
Has any of this changed recently?
Assurance of future supply may be at risk due to supply realignments; reputable suppliers support
full transparency and will be forthcoming about the impact of any changes.
Can we make a site visit? Perform an audit?
Demonstrates transparency and standards compliance, provides opportunity to validate supplier
claims; go where you want to go, see what you want to see.
Discussion Point #2
Integrity of Supply
Integrity of supply means that all aspects
of product quality and traceability are
well-documented, validated by
independent audit, and completely
transparent. In their recent article, Siegel
and Foster1 emphasized the importance
of “exercising extra vigilance in confirming
the integrity and authenticity” of
information provided by a supplier and
performing “due diligence in vendor
qualification of all serum suppliers.”
They encouraged sera users to do a
“thorough audit of the traceability
system,” to “know your vendors,” to
conduct “proper and periodic on-site
audits,” and ask for the appropriate
“credentials.” These recommendations
underscore the fact that strategic,
quantifiable differences exist
between suppliers, their products,
and their operations.
The International Serum Industry
Association (ISIA) has established
industry standards and certification
programs to aid sera users in
substantiating integrity of supply.4 Five
years ago, the ISIA developed and
implemented a rigid program of
independent audits to verify
compliance with traceability standards.
4
VWR International I VWRbioMarke Issue 1 I April 2015
Elite status as an ISIA Traceability
Certified supplier is awarded to those
who demonstrate full compliance
with ISIA guidelines and are the subject
of a successful audit. Further, it has
established strict guidelines for product
quality testing and reporting on
documents like certificates of analysis
(CoA). Sera users should source
exclusively from ISIA-certified
companies to ensure traceability and
product quality. A list of certified
suppliers, filtration partners, and raw
material providers is maintained on the
ISIA’s website.6
Product quality and traceability is also
enhanced by validated technology
enhancements in the manufacturing
process. Implementation of single-use,
disposable filtration technology
eliminates cross-contamination risk
from lot-to-lot and maintains true
traceability — a technology that is widely
used downstream in bioproduction
environments. Additional measures such
as maintaining the cold chain during
filtration ensures that the bioburden
of the sera is unchanged during
processing and final packaging.
Sera users should use the above standards,
programs, and technologies to
CeLL Biology
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Integrity of Supply
What Questions to Ask
Why is it Important
Is your entire supply chain ISIA Traceability Certified (raw material collection, processing,
filtration, fulfillment)?
Identifies suppliers you can trust; addresses the issues that prompted mass product recalls.
Is your product documentation ISIA compliant (CoA)?
Demonstrates commitment to the most relevant quality and tracebility standards.
Is your product testing done in-house or by independent labs?
Ensures transparency and accuracy.
Have you implemented validated technology enhancements in your manufacturing
process?
Eliminates cross-contamination from lot-to-lot and maintains true traceability.
Do you maintain the cold chain during manufacturing?
Minimizes bioburden and endotoxin contribution of all processes.
Has any of this changed recently?
Identifies effort to comply with standards or exposes inability to comply
Can we make a site visit? Perform an audit?
Demonstrates transparency and standards compliance, provides opportunity to validate supplier
claims; go where you want to go, see what you want to see.
comprehensively examine a supplier’s
integrity of supply, conduct on-site audits,
and identify and discuss any points of
non-compliance. Any hesitation in this
regard on the part of a supplier is a serious
cause for concern.
Conclusion
Changing market dynamics have altered
the historical paradigm for sourcing sera.
Sera suppliers may be hesitant to explore
the impacts of these dynamics with sera
users as it exposes problem areas that, to
this point, were overlooked or ignored.
However, the exercise serves the long-term
interests of both sera suppliers and users.
The responsibility to scrutinize the supply
strength and product integrity of a supplier
rests squarely on the shoulders of sera
users. The discussion points outlined in this
article will facilitate sera users in the
discharge of that responsibility and lead to
a stronger, long-term relationship with
their ideal sera supplier.
Ultimate Grade
About the Authors
Devin Davis is the founder and Vice President of
Seradigm, now part of VWR International. Prior to
founding Seradigm, Mr. Davis held various
positions over several years at Thermo Fisher
Scientific supporting the HyClone brand. Sherrie
Drake Hirschi is Marketing Manager for Seradigm,
now part of VWR International. Previously she
spent nine years at HyClone and was HyClone’s
Serum Product Manager for seven years.
References
1. Siegel W, Foster L. Fetal bovine serum: the impact of geography.
BioProcess J, 2013; 12(3): 28-30.http://dx.doi.org/10.12665/
J123.Siegel
2. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfRes/res.
cfm?ID=117863
3. The World Organisation for Animal Health (OIE). OIE BSE Risk
Status. http://www.oie.int/en/animal-health-in-the-world/
official-disease-status/bse/list-of-bse-risk-status/
4. International Serum Industry Association (ISIA).
http://www.serumindustry.org.
5. United States Department of Agriculture (USDA).
http://www.nass.usda.gov/Surveys/Guide_to_NASS_Surveys/
Cattle_Inventory/index. asp ; http://usda01.library.cornell.edu/
usda/current/Catt/Catt-01-31-2014.txt.
6. International Serum Industry Association (ISIA).
http://www.serumindustry.org/traceability.htm.
Premium Grade FBS
USDA Approved Origin FBS
Size
Additional
Treatment
50mL
None
89510-198 50mL
None
89510-194 50mL
None
89510-182
50mL
Heat Inactivated
89510-200 50mL
Heat Inactivated
89510-196 50mL
Heat Inactivated
89510-184
500mL
None
97068-101 500mL
None
97068-085 500mL
None
89510-186
500mL
Heat Inactivated
97068-107 500mL
Heat Inactivated
97068-091 500mL
Heat Inactivated
89510-188
Cat. No. Size
Additional
Treatment
Cat. No. Size
Additional
Treatment
Cat. No.
Gamma irradiation available upon request.
April 2015 I VWRbioMarke Issue 1 I VWR International
5
Corning® Cytokines, Growth Factors,
and Other Media Supplements
 For proliferation or differentiation of the affected cells
 Immunology and oncology research
 Serum-free supplementation and sera replacements
Our portfolio of cytokines, growth factors,
and media supplementation offers a wide
array of proteins that result in proliferation or
differentiation of the affected cells.
Cytokines are a class of signaling molecules or
intercellular mediators (proteins, peptides, and
glycoproteins) that primarily affect cells of the
immune system; however, they can affect
various cell types outside of the immune
system. They differ from classical hormones in
that they are produced by a number of tissue or
cell types rather than by specialized glands. The
effects of cytokines on cells can yield different
outcomes. Some cytokines cause cell
proliferation, while others may cause
chemotaxis between cell types, and others can
even cause cell death. Cytokines and growth
factors are similar in their structure and
mechanism of action. Both bind and initiate
signaling pathways and many share several
intracellular signaling components.
Growth factors are proteins that bind to
receptors found on the surface of
6
VWR International I VWRbioMarke Issue 1 I April 2015
non-hematopoietic cells. Each family of growth
factors affects specific cell types, e.g., nerve
growth factors (NGF) affect nerve cell types,
and epidermal growth factors (EGF) affect
epithelial cell types.
These products provide researchers in
academia and the pharmaceutical industry
with research tools as the need for defined,
serum-free media grows. Selecting the
appropriate growth factors and cytokines for
your application is important – this is often
based on existing protocols, experience, or
experimentation. The mechanism of action
of some growth factors is still heavily
researched. Some cell lines depend on
certain supplementation – whether that
be a general growth supplement or a
pinpointed growth factor – to differentiate
or proliferate.
CeLL Biology
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Corning Endothelial Cell Growth
Supplement (ECGS)
wide variety of cell types under serum-free
or reduced serum conditions.
Corning ECGS is a broadly used supplement
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endothelial cells. ECGS contains various
growth factors (e.g., acidic FGF or ECGF-α).
Corning Interleukin-2 (IL-2), Human
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Corning Epidermal Growth
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Grade)
Corning EGF, Mouse Natural, is a
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Corning Epidermal Growth
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Corning EGF, Human Recombinant, is a
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Corning Basic Fibroblast
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Human Recombinant
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Corning IL-2, Human Recombinant,
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under reduced serum conditions.
* ITS and ITS+ differ in formulation, state, and
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Corning T-Cell Culture
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Description
Size
Endothelial Cell Growth Supplement (ECGS)
15 mg
62405-784
Cat. No.
Endothelial Cell Growth Supplement (ECGS)
100 mg
47743-652
Epidermal Growth Factor (EGF), Human Recombinant
100 μg
47743-566
Epidermal Growth Factor (EGF), Mouse Natural (Culture Grade)
100 μg
47743-566
Basic Fibroblast Growth Factors (bFGF), Human Recombinant
10 μg
47743-574
Interleukin-2 (IL-2), Human Recombinant
10,000 BRMP Units
47743-742
IL-3 Culture Supplement, Mouse
25 mL
47743-646
ITS Premix Universal Culture Supplement
5 mL
47743-626
ITS+ Premix Universal Culture Supplement
20 mL
47743-628
T-Cell Culture Supplement with ConA (IL-2 Culture Supplement), Rat
100 mL
47743-642
April 2015 I VWRbioMarke Issue 1 I VWR International
7
siRNA Dependent Gene Silencing in HeLa Cells
Cultivated on Various Cell Culture Surfaces
Introduction
Methods
RNA interference (RNAi)
Transfection of HeLa
Cells with siRNA
In the following experiment, HeLa cells
were transfected with Cy®3-conjugated
GAPDH (Glyceraldehyde-3-phosphate
dehydrogenase) siRNA on Tissue Culture
(CellStar™ TC), Advanced TC™ (AdvTC),
Poly-D-Lysine (PDL) and Collagen Type I
surfaces to evaluate the impact of the
cultivation surface on the siRNA
transfection efficiency. This correlation was
already proved for transfection with
pcDNA3 plasmid encoding GFP (Green
fluorescent protein) and luciferase
respectively in HEK293 cells in
former studies.
Brightfield
FLUOrescence
E
B
F
ADVNACED TC
TC-Treated
A
COLLAGEN Type 1 PDL Coated
0.3μL of 10μM of GAPDH and negative
control siRNA was pipetted in triplets into
the wells of the 96 well microplates (Tissue
Culture, Advanced TC, Poly-D-Lysine and
Collagen Type I surface). Using a sterile
tube, a dilution of 0.5μL transfection agent
and 19.5μL OptiMEM medium per well was
prepared, incubated for 10 min and
pipetted onto the siRNA. After 10 min of
incubation at room temperature, 80μL of
cell suspension containing 4,000 cells were
pipetted onto the siRNA and incubated for
48h at 37°C in the tissue culture incubator.
GAPDH Assay
C
D
G
H
Figure 1: Transfection of Cy3-labeled GAPDH-siRNS
in HeLa calls plated on tissue culture (A,E), Advanced
TC (B,F), Poly-D-Lysine (C,G) and Collagen Type I
microplates (D,H). Brightfield images demonstrate
the morphology of plated cells (A-D) while
fluorescence pictures show the transfection rate of
Cy3 labelled siRNA on different surfaces (E-H).
8
A reverse high-throughput screening
transfection protocol was used to introduce
siRNA into HeLa cells. First, siRNA was
pipetted into the wells, followed by
complexation with transfection reagent.
Finally, siRNA complexes were overlaid
with cells. Cells were trypsinized before
transfection using the standard procedure
and resuspended in growth medium
(Earle’s MEM, 10% FCS, 2% Glutamine, 2%
non-essential amino acids) at a density of
50,000 cells/mL.
GAPDH expression serves as a marker for
cellular toxicity resulting from transfection.
Putative cytotoxic effects can be identified
by transfecting cells with negative control
siRNA and evaluating whether the
transfection leads to a decrease in
endogenous GAPDH protein levels.
For the detection of GAPDH, media was
removed from the cells and replaced with
200μL of lysis buffer. After 20 min
incubation at 4°C, the lysate was
homogenized by pipetting up and down.
10μL were then transferred to a new
transparent 96-well microplate. 90μL of
freshly prepared substrate were pipetted
onto the cell lysate. Immediately
VWR International I VWRbioMarke Issue 1 I April 2015
afterwards, a kinetic readout was
performed for a period of 3 minutes using
a fluorescence reader at 560/590nm. The
activity of GAPDH was calculated by
subtracting the fluorescence at t0 min
from the endpoint measurement at t3 min.
Results
Transfection Efficiency in HeLa Cells
Cultivated on Various Surfaces
Detected by Cy3 Labeled siRNA
Cy3 labeled GAPDH siRNA was transfected
in HeLa cells cultivated on tissue culture
(TC), Advanced TC, Poly-DLysine and
Collagen Type I microplates according to a
high throughput screening (HTS) protocol.
The transfection efficiency was detected
based on Cy3 driven fluorescence via
fluorescence microscopy after 48h. On all
surfaces, a confluent monolayer of HeLa
cells could be observed after transfection
(Fig. 1 A-D). According to the fluorescence
signal obtained using Cy3 labelled siRNA,
an efficient transfection of GAPDH siRNA
into HeLa cells occurred with all tested
surfaces (Fig. 1 E-H). A comparison of the
microscopic images obtained with the
different cultivation surfaces indicates that
the transfection was most efficient with
cells cultivated on the Advanced TC and
Poly-D-Lysine surfaces. However,
fluorescence was also detected with all
other surfaces.
Influence of the Cultivation Surface
on GAPDH mRNA Silencing
The GAPDH protein knockdown of siRNA
transfected HeLa cells was evaluated using a
fluorescence based assay. Substrate formed
by the oxidation of NAD+ to NADH in the
presence of G-3-P and phosphate by GAPDH
was quantified based on its fluorescence at
560/590nm over a 3 minute interval.
GAPDH siRNA transfected cells were
compared to cells transfected with a
negative control siRNA and nontransfected
cells. All transfections were conducted on
Tissue Culture, Advanced TC, Poly-D-Lysine
CeLL Biology
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Figure 2: Evaluation of GAPDH knockdown by
RNAi technology of HeLa cells plated on various
surfaces. HeLa cells were transfected with siRNA
against GAPDH mRNA and a negative control siRNA
construct to show the influence of the surface on
GAPDH protein knockdown. Non-transfected HeLa
cells were used as a control to evaluate cytotoxicity
of transfection itself. THE GAPDH activity was
calculated by the increase of fluorescence at
560/590nm at t0 min and t3 min.
Figure 3: Graphic chart of the total GAPDH
knockdown of HeLa cells transfected with GAPDH
siRNA on Tissue Culture, Advanced TC, Poly-D-Lysine
and Collagen Type I surfaces.
Figure 4: Cell viability of siRNA transfection in HeLa
cells cultivated on different surfaces.
with negative control siRNA, cytotoxic
effects of the transfection procedure
could be assessed.
and on Collagen Type I microplates,
respectively (Fig. 2).
Cell viability of transfected HeLa cells
varied from 83.8% when cultured on
standard tissue culture surface, to 87.7%
on Advanced TC surface, 87.0% on
Poly-D-Lysine and 92.4 % on Collagen Type
I surface (Fig. 4).
tissue culture and Collagen Type I surfaces
also performed well in the described
experiment. Cytotoxic effects were further
evaluated by comparing non-transfected
HeLa cells to cells transfected with negative
control siRNA. In general, the cytotoxic
effects of transfection were quite low on all
tested surfaces. In summary, all tested cell
culture surfaces were suitable for siRNA
transfection in HeLa cells. An enhancement
of transfection efficiency and protein
knockdown was achieved with the use of
the Poly-D-Lysine and the Advanced
TC surface.
GAPDH knockdown was evaluated by
comparing cells transfected with negative
control siRNA and cells transfected with
GAPDH siRNA. GAPDH knockdown was
observed on all tested surfaces.
Since the total knockdown reached 55.3%
for cells cultivated on the standard tissue
culture surface, siRNA dependent protein
knockdown was increased by 24% to
achieve a total knockdown of 79.3% for cells
cultivated on the Advanced TC surface.
Similar results were obtained with the
Poly-D-Lysine surface, where siRNAdependent protein knockdown was
increased by 23.1% to achieve 78.4% in
total. Cells cultivated on Collagen Type I
showed a 69.6% total GAPDH knockdown
response to siRNA, an increase of 14.3% as
compared to the standard TC surface (Fig. 3).
Cytotoxic Effects of siRNA
Transfection on HeLa Cells
Cultivated on Different Surfaces
By comparing GAPDH activity in nontransfected controls with cells transfected
Conclusion
The influence of the culture surface on the
efficiency of siRNA transfection and
corresponding protein knockdown was
evaluated in the performed experiment. For
that reason, GAPDH siRNA as well as
negative control siRNA was transfected in
HeLa cells cultured on standard tissue
culture, Advanced TC, Poly-D-Lysine and
Collagen Type I surfaces. Both transfection
efficiency, measured using Cy3 labelled
siRNA with fluorescence microscopy,
as well as corresponding GAPDH
knockdown, evaluated by protein assay,
were determined to be the most
effective when using Advanced TC and
Poly-D-Lysine surfaces.
Although the results indicate that these
surfaces are the most suitable ones for
siRNA transfection in HeLa cells, standard
Description
Cat. No.
Advanced TC Plates with Lids, Sterile
6-Well Plate, Clear
89131-688
12-Well Plate, Clear
89136-864
24-Well Plate, Clear
89131-690
48-Well Plate, Clear
89136-866
96-Well Plate, White/Clear
89136-852
96-Well Plate, Black/μClear
89131-694
384-Well Plate, Black/μClear
89131-696
CELLSTAR TC Treated Plates with Lids, Sterile
6-Well Plate, Clear
82050-842
12-Well Plate, Clear
82050-930
24-Well Plate, Clear
82050-892
48-Well Plate, Clear
82051-004
96-Well Plate, White/Clear
82050-758
96-Well Plate, Black/μClear
82050-748
384-Well Plate, Black/μClear
82051-282
April 2015 I VWRbioMarke Issue 1 I VWR International
9
FectoPRO
TM
Achieve Amazing Protein Yields in CHO and HEK-293 Cells
Transient gene expression (TGE) is commonly used for
medium scale production of recombinant proteins
and antibodies. This approach allows generation of
sufficient protein amounts avoiding a major investment in
production of stable cell lines prior to “proof of concept”
studies or tools validation. Indeed, the speed and
flexibility of TGE has enabled this technique to be widely
Reagent
FectoPRO™
+ FectoPRO™
Booster
Competitor A
Reagent
DNA
amount µg/
mL cell
culture
Reagent
volume µL/
mL cell
culture
0.4 - 0.6
1.25
0.6 - 0.9
1.25
Table 1. Amount of DNA and volume of
reagent needed for transfection according
to manufacturers’ recommendations.
Figure 1. FectoPRO
outperforms the competitor
reagents and achieves the
highest transfection efficiency.
Competitor A CHO-S and
293-F cells were seeded at
1 x 10 6 cells/mL in 30mL of
their recommended media
and transfected using a GFP
expressing plasmid with
Competitor A reagent
(1.25 µg/mL), Competitor B
(1µg DNA/mL), or FectoPRO +
FectoPRO Booster (0.5 µg/mL).
GFP expression was measured
24h after transfection using
fluorescence microscopy.
adopted in bioproduction for early discovery, research
applications and process developments. However, the
protein yields usually remain lower with TGE than with
stable gene expression process. In order to improve
productivity, Polyplus-transfection has developed a novel
transfection kit to generate superior protein amounts by
TGE for medium scale bioproduction.
Introduction
Methods and Results
FectoPRO is a novel, powerful solution for
improved protein yields in TGE systems. This
kit was designed after extensive screening
of numerous chemical structures based on
their transfection efficiency, protein
production yield, and cell viability. It is
suitable for transfection of suspension CHO and
HEK-293 cells in various serum-free media using
low DNA amounts (<1µg/mL of cell culture).
Transfection Efficiency
FectoPRO outperforms other currently available
transfection reagents such as PEIs or lipidbased reagents and shows unprecedented
results in both CHO and HEK-293 cells.
Competitor A
Competitor B
We compared FectoPRO with other commercially
available transfection reagents and measured
the obtained transfection efficiency. FectoPRO in
combination with its booster gave by far the
highest transfection efficiency in both
suspension CHO and HEK-293 cells (Fig.1).
Protein Yields
Productivity was assessed by production of
an IgG fragment. In both CHO and HEK-293
cells, FectoPRO largely outperforms
other transfection reagents commonly
used in bioproduction processes in terms of
protein yields (Fig.2).
FectpPRO+ Booster
CHO
Competitor A
Competitor B
FectpPRO+ Booster
HEK-293
10
VWR International I VWRbioMarke Issue 1 I April 2015
CeLL Biology
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Cost Effective Transient
Gene Expression
High quality plasmid DNA preparation
can be very costly, especially when large
DNA amounts are required for large scale
protein production. FectoPRO saves on
DNA cost by using only 0.4 to 0.6µg
DNA/106 cells/mL of cell culture making
this kit an affordable and economical
solution for bioproduction processes.
CHO
Highly Scalable
The FectoPRO-mediated transfection
process is easily scalable from a few
milliliters to several liters of cell culture,
ensuring robust reproducible protein
production. FectoPRO simplifies your
bioproduction process with a robust
protocol that is easily adapted to different
culture vessels (Fig.3).
HEK-293
Figure 2. FectoPRO leads to unprecedented protein yields in TGE. CHO and HEK-293 cells were
transfected with a IgG3-Fc expressing plasmid using reagents and conditions mentioned. Antibody
quantitation was performed by using protein G affinity column (HPLC) and qualitative analysis was done
by Western Blot 120 hours post transfection.
CHO
Compliant with Biomanufacturing
Guidelines
FectoPRO is chemically defined and
guaranteed free of animal-origin
components. Systematic lot management
and release testing is performed for each
lot produced. FectoPRO transfection kit
undergoes advanced quality controls for
protein productivity, cell viability, and
complete sterility.
HEK-293
In addition, Polyplus-transfection is ISO
9001 Quality Management System
accredited since 2002. This level of
certification ensures our customers
worldwide that we have established
reliable and effective processes for product
development, manufacturing, sales, and
customer support.
Figure 3. FectoPRO is easily scalable and reproducible. IgG3-Fc production in different volumes of cell
culture after transfection with DNA/FectoPRO (0.4µg/0.72µL) and Booster (0.25µL) per mL of CHO cell
culture and with DNA/FectoPRO (0.5µg/0.75µL) and Booster (0.30µL) per mL of HEK-293 cell culture.
Quantitation was performed by using protein G affinity column (HPLC).
Volume, mL
Cat. No.
1
10118-842
10
10118-444
April 2015 I VWRbioMarke Issue 1 I VWR International
11
Understanding the Molecular
Basis of Parkinson’s Disease:
Cell-based
Model of
Mitophagy and
Aggresome
Accumulation
ABSTRACT
Parkinson’s disease (PD) is the 2nd most
common neurodegenerative disorder with
over 6 million cases worldwide. Data from
various studies of genes associated with
hereditary disease, toxicology studies using
animal models/in vitro models and also
patient-based studies have implicated
compromised protein turnover relating to
the ubiquitin-proteasome system (UPS)
and autophagy-lysosome pathway (ALP),
as well as diminished mitochondrial
activity in the most common idiopathic
forms of the disease. Mutations in the
Parkin gene are a primary cause of
autosomal recessive juvenile PD. Parkin
functions as an E3 ligase that
ubiquitinylates α-synuclein, the primary
aggregated protein associated with the
neurotoxic accumulation in Lewy bodies.
Parkin also interacts with and
ubiquitinylates depolarized mitochondria,
promoting their clearance through
mitophagy. Cell-based assays relevant to
monitoring aspects of PD are presented,
including aberrant aggresome formation,
mitochondrial depolarization,
ubiquitinylation, and mitophagy. The
highlighted assays contribute to the
understanding of regulatory pathways
controlling mitophagy and Lewy body
formation, aiding in the characterization of
12
various human neuropathological
disorders, including PD.
BACKGROUND
Many genes mutated in hereditary PD
play a pathogenic role in mitochondria as
well as in the ubiquitin-proteasome
system (UPS) and the autophagylysosome pathway (ALP), both of which
dispose of misfolded proteins and
damaged organelles. α-Synuclein
association with the inner mitochondrial
membrane coincides with selective
age-related mitochondrial complex I
inhibition and decreased respiration,
along with increased mitophagy.
α-Synuclein is the major misfolded
protein of Lewy bodies, a fundamental
pathological feature of the
degenerating PD brain.
Role of Ubiquitin in
Parkinson’s Disease
Parkin is a RING-type E3 ubiquitinprotein ligase that triggers selective
ubiquitinylation and targeting of
depolarized mitochondria for
sequestration in aggresomes and/or
autophagosomes, leading to degradation
by the ALP. Parkin ubiquitinylates Hsp70
on multiple residues. Lewy bodies are
positive for molecular chaperones,
VWR International I VWRbioMarke Issue 1 I April 2015
suggesting they play a role in progression
of PD. Interference with chaperone
activity accelerates α-synuclein toxicity.
α-Synuclein- and ubiquitin-positive
inclusion bodies are the pathological
hallmarks of PD.
Detecting autophagy, mitochondrial
degradation, and the formation of
aggresomes and inclusion bodies are
relevant to the monitoring of different
aspects of PD and important tools to aid in
PD drug discovery.
CONCLUSIONS
• The pathophysiology of PD is intricately
entwined with ubiquitinylation through
the UPS and ALP.
• Various CELLestial® dyes can readily be
implemented in phenotypic assays
targeting different aspects of PD.
• The phenotypic assays could potentially
be used to identify new drugs that
selectively target PD-associated E3
ubiquitin-protein ligases, such
as parkin.
References
1. J. Sutcliffe et al. Neurosci Res. (2011) 89 808
2. D. Shen, et al. Cell Biochem and Biophys (2011) 60 173
3. I. Raju et al. PLoS One (2011) 6(5) e19876
4. S. Sarkar et al. Nat. Chem. Biol. (2007) 3 331
5. M. Renna et al. J. Biol. Chem. (2010) 285 11061
CeLL Biology
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Phenotypic Assays Relevant to Screening of Small Molecules in PD Drug Discovery
Autophagy: Cyto-ID® Autophagy Detection Kit (Fig. 1)
Mitochondrial Degradation: Mito-ID® Red Detection
Kit (Fig. 2)
Figure 1: Cyto-ID® Autophagy Detection Dye (green) signal increases about 2.5-fold after 1 hr starvation.
Nuclei were counterstained with Hoechst 33342 (blue).
Figure 2: Cells were stained with Mito-ID® Red dye
for 15 minutes. Nuclei were counter-stained with
Hoechst 33342 dye.
Aggresomes, Inclusion Bodies & Lewy Bodies: ProteoStat® Aggresome Detection Kit (Fig. 3)
Figure 3 (Left): Aggresomes with HeLa cells, treated
for 12 hours with 5µM proteasome inhibitor MG-132
(right), detected by ProteoStat® Aggresome dye (red)
and counterstained with Hoechst 33342.
A
B
C
D
Fig.4 (Left): Parkin interacts with and ubiquitinylates
depolarized mitochondria, promoting their clearance
by mitophagy; aggresomes accumulate within cells
undergoing mitophagy.
Figure 4: A) Co-localization of parkin and mitochondria is evident. HeLa cells were transfected with Parkin for 1 hr
and then treated with DMSO (control) or 10µM CCCP, an inducer of mitochondrial dysfunction for 1 hr. The cells
were fixed, permeabilized, and stained with Alexa Fluor 488 dye-labeled parkin Ab, then followed by Mito-ID®
Red dye. B) Parkin promotes mitochondrial ubiquitinylation following CCCP treatment. Parkin-transfected HeLa cells
were treated with DMSO (control) or 10µM CCCP for 24 hr. The cells were fixed and immunostained using Atto
488 dye-labeled ubiquitin antibody and Mito-ID® Red dye. C) Parkin induces elimination of de-energized
mitochondria by mitophagy. Parkin-transfected HeLa cells were treated with DMSO (control) or 10 μM CCCP
for 24 hr. The cells were incubated with Cyto-ID® Green, Mito-ID® Red and Hoechst dye for 15 min at 37°C.
D) Aggregated proteins coalesce in peri-nuclear Lewy body-like structures within cells undergoing mitophagy.
Composite bright-field and fluorescence microscopy images: Parkin-transfected HeLa cells were treated with
DMSO (control) or 10 μM CCCP overnight. The cells were fixed and permeabilized, then stained with Hoechst
dye and ProteoStat® Aggresome Detection Dye.
Description
Cat. No.
Cyto-ID®Autophagy
Detection Kit
89165-926
Mito-ID® Red Detection Kit
89165-876
ProteoStat® Aggresome Detection Kit
89409-190
April 2015 I VWRbioMarke Issue 1 I VWR International
13
Intrawell Cell Distribution
in Nunc® Microwell Plates
Peter Esser, Senior Scientist, and Louise Gjelstrup, Laboratory Technician
Thermo Scientific Laboratories
It is a well known fact that temperature gradients and vibrations
during cell settlement in cell culture flasks and plates may cause uneven
cell distribution patterns on the growth surfaces1, 2. Therefore, all
ingredients assembled should be left in absolute tranquility (i.e., no
temperature gradients, no vibrations, and no ventilation) during cell
settlement. As communicated elsewhere3, this is most easily accomplished
by pre-incubation of the seeded culture at room temperature (RT).
The significance of the evaporation reservoir in the Thermo Scientific™ Nunc
Edge Plate in relation to the uneven cell distribution was investigated with either
200 or 100µL MDCK cell suspension per well according to the following 4-plate test set-up
distinguishing four different situations framed in red:
Plate Number
Conditions
1
2
3
4
Plate
RT
RT
RT
RT
Cell Suspension
RT
RT
RT
RT
RT water*
RT water*
Empty
Empty
Pre-incubation
Reservoir
none
2 hrs at RT
none
2 hrs at RT
Incubation**
37°C
37°C
37°C
37°C
* 1.75 mL per reservoir compartment
**5% CO2 in air
Figure 1.
Edge Plates
seeded with
200µL MDCK
suspension per
well: Stained
with crystal
violet after
incubation at
37°C for 3 days.
14
Plate 1 - Reservoir filled, no pre-incubation
Plate 2 - Reservoir filled, pre-incubation
Plate 3 - Reservoir empty, no pre-incubation
Plate 4 - Reservoir empty, pre-incubation
VWR International I VWRbioMarke Issue 1 I April 2015
CeLL Biology
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Figure 1 shows the results with 200µL cell
suspension per well, where it is seen that
without pre-incubation (Plates 1 and 3)
outward “half-moon” cell accumulations
occur in the edge wells, but to a lesser
degree in the plate with the reservoir filled
(Plate 1) compared to the plate with an
empty reservoir (Plate 3). In the latter case,
with an empty reservoir and no preincubation, additional patterns occur in the
edge wells, which may stem from
incubator vibrations. However, both
phenomena are eliminated by preincubation (Plates 2 and 4). Therefore, the
reservoir content may to some extent act
as a “buffer” against uneven cell
distribution in the (edge) wells but has no
significance if pre-incubation is employed.
Figure 2 shows the results with 100µL cell
suspension per well, where edge effects
are largely absent, thus indicating a
volume-dependent reverse effect. This
may be explained by the shorter settling
distance and time, making the cell
distribution less sensitive to thermal
disturbances in the wells.
Figure 3 theoretically explains the cell
distribution skewing, observed with 200µL
cell suspension, by temperature gradients
upon incubation at 37°C. In model experiments with suspended, descending particles
in water it has been demonstrated that a
convection stream circulating as illustrated
would indeed occur when heating the side
of the vessel, and it would “sweep” the
particles into the heated corner of the vessel.
References
1. Nielsen V. and Esser P. Incubator Shelf “Images” in Monolayer
Culture. Nunc Bulletin No. 3, 2nd Ed. 1997.
2. Nielsen V. Vibration Patterns in Tissue Culture Vessels. Nunc
Bulletin No. 2, 2nd Ed. 1997.
3. Lundholt B. K. et al. A Simple Technique for Reducing Edge Effect in
Cell-Based Assays. Journal of Biomolecular Screening 8(5), 2003.
Description
Cat. No.
Surface Treated, Sterile
89131-504
Untreated, Sterile
89131-508
Untreated, Non-sterile
89131-506
Plate 1 - Reservoir filled, no pre-incubation
Plate 2 - Reservoir filled, pre-incubation
Plate 3 - Reservoir empty, no pre-incubation
Plate 4 - Reservoir empty, pre-incubation
Figure 2. Edge Plates seeded with 100µL MDCK suspension per well: Stained with crystal violet after
incubation at 37°C for 3 days.
Plate 1 - Reservoir filled, no pre-incubation
Plate 2 - Reservoir filled, pre-incubation
R
R
S
Plate 3 - Reservoir empty, no pre-incubation
Plate 4 - Reservoir empty, pre-incubation
R
R
S
Figure 3. Theoretical profile scenarios in Nunc Edge Plates with the reservoir (R) filled (top) or with an
empty reservoir (bottom), when put on shelves (S) in 37°C incubator immediately after addition of cell
suspension at RT (left), or after pre-incubation with cell suspension at RT (right). The uneven heating at
the plate edges will create circulating convection streams in the edge wells (as opposed to the central
wells having more homogeneous convection). Without pre-incubation, descending cells accumulate at
the outward parts of the well corners (pink arrows) to a larger degree with empty reservoir than with the
“buffering” effect of filled reservoir. With pre-incubation, where the cells have been allowed to settle in
the absence of temperature gradients, convection streams will not skew the cell distribution pattern. A
denser rim of cells all around the well corners (blue arrows) will always be observed reflecting the larger
liquid column heights (thus more cells) at the well periphery than at the center.
April 2015 I VWRbioMarke Issue 1 I VWR International
15
Unique Enzymes from the Arctic
Alleviate the Need to Purify
Samples for Several Applications
Unique recombinant enzymes derived from various arctic species are adapted to cold temperatures and are
well-suited for standard life science applications, with the added convenience and efficiency of irreversible heat
inactivation. Unlike their standard counterparts, these cold-adapted enzymes can be completely inactivated via a
high temperature inactivation step, eliminating any requirement for downstream sample purification.
Figure 1A
Figure 1B
Figure 1.Shrimp Alkaline
Phosphatase, Recombinant
(rSAP) treatment reduces empty
vector background in molecular
cloning. Linearized pUC19 was
treated with rSAP or water
prior to ligation with an insert.
The rSAP was irreversibly
heat-inactivated and the
samples were used without
any purification steps before
the ligation reaction.
Heat Inactivation
100
75ºC
% Activity
80
65ºC
60
40
20
0
0
2
4
6
8
10
Minutes
Shrimp Alkaline Phosphatase,
Recombinant (rSAP)
VWR Life Science AMRESCO offers Shrimp
Alkaline Phosphatase, Recombinant (rSAP),
which nonspecifically dephosphorylates
the 5’ ends of nucleic acids. This activity
streamlines molecular cloning by
preventing self-ligation of linearized
plasmid DNA, thus ensuring low vector
background for colony selection. Plasmid
DNA may be treated with rSAP
concurrently with restriction digestion or
by a short, 10 minute incubation postdigestion. The rSAP is then irreversibly
inactivated by heating to 65°C for 5
minutes, after which the linearized
plasmid may be used directly in a ligation
reaction. Other useful applications for
rSAP include 5’ end-labeling, for which
it facilitates the replacement of
unlabeled phosphates with labeled
phosphate groups, and PCR cleanup
prior to DNA sequencing or SNP analysis.
16
The latter application is performed
by combining the nucleotide
dephosphorylating activity of rSAP
with the primer degrading activity of
Exonuclease I. This effectively removes
the ability of unincorporated
nucleotides and primers from a PCR
reaction to interfere with the primer
extension reaction for sequencing
or genotyping.
To demonstrate the effectiveness of rSAP
activity in molecular cloning, pUC19
digested with SmaI was treated with rSAP
or water and then ligated with a 1kb insert.
The ligation products were transformed
into competent E. coli and plated in
triplicate on LB medium containing
ampicillin and VWR Life Science
AMRESCO’s X-Gal/IPTG Ready Solution for
blue-white colony screening. Blue and
white colonies were compared among the
plates for each condition. The number of
VWR International I VWRbioMarke Issue 1 I April 2015
blue colonies, which represent empty
vectors, was significantly lower for the
plates transformed with rSAP-treated
samples than for those that were
untreated (Figure 1A). Plasmids isolated
from several white colonies on each
plate were confirmed by restriction
analysis to contain the expected insert
(data not shown). These data indicate
that rSAP treatment effectively
decreased the number of self-ligating
vectors prior to ligation with insert,
and that rSAP was completely inactivated
by heat treatment, allowing ligation
between vector and insert with intact 5’
phosphate to proceed. A separate
experiment measuring rSAP activity with a
substrate by spectroscopy further
confirmed the efficiency of heat
inactivation. After only a 5 minute
incubation at 65°C or 1 minute at 75°C
there was no detectable rSAP activity
(Figure 1B).
Genomics
Uracil-DNA Glycosylase (UNG), Cod
Highly sensitive PCR and RT-qPCR have
become routine assays in many life science
labs, but they are unfortunately vulnerable
to contamination from environmental
DNA, and more significantly, to previous
PCR products. Carryover PCR
contamination can cause false positive
results, especially in labs continuously
amplifying a particular set of targets.
Guidelines for prevention of
contamination, such as physical separation
of sample preparation and amplification
areas, have been well-documented and are
followed in most high-throughput labs.
As added precaution, it is also possible to
specifically degrade carryover PCR
products using enzymatic digestion.
VWR Life Science AMRESCO’s Uracil-DNA
Glycosylase (UNG), Cod is a thermolabile
recombinant enzyme that degrades
uracil-containing single- and doublestranded DNA, but not RNA or thymidine
containing DNA. The degradation occurs
upon exposure to alkaline conditions and
high temperature at sensitive sites
generated by the hydrolysis of the
N-glycosidic bond between deoxyribose
sugar and the base in uracil. The
prerequisite for UNG pre-treatment of PCR
reactions is that dUTP be substituted in
place of dTTP during all amplification
reactions, such that all PCR amplicons can
become substrates for UNG and all dTTP
DNA will not.
In contrast to competing Uracil-DNA
Glycosylases, VWR Life Science AMRESCO’s
arctic-derived UNG has the distinct
advantage of having a low inactivation
temperature of 45°C, which enables
carryover decontamination of one-step
reverse transcription reactions with UNG
prior to the cDNA synthesis step.
Inactivation is irreversible after heating just
20 minutes at 55® or 1 second at 95°C,
allowing for stability of the cDNA that has
incorporated dUTP. The irreversible
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
inactivation feature of VWR Life
Science AMRESCO UNG also facilitates
greater downstream manipulation and
stability of newly synthesized dUTPcontaining DNA by PCR, allowing for
long-term storage, restriction digestion,
cloning, sequencing and hybridization
applications. The gels in Figure 2
demonstrate the effective elimination of
dUTP-containing DNA from spiked PCR
samples that were treated or untreated
with UNG for 5 minutes prior to PCR. The
stability of the newly synthesized dUTPcontaining amplicons after irreversible
heat inactivation of UNG was assessed by
gel analysis of the treated and untreated
PCR samples after storage at room
temperature for four days.
DNase, Double-Strand
Specific, Heat-Labile
VWR Life Science AMRESCO’s DNase,
Double-Strand Specific, Heat-Labile is an
arctic-adapted recombinant endonuclease
that cleaves phosphodiester bonds in DNA
to yield 2–8bp oligonucleotides with
5’-phosphate and 3’-hydroxyl termini. The
highly specific activity of this enzyme
toward dsDNA can be inactivated by
heating at 55°C, conveniently eliminating
the need for its physical or chemical
removal before downstream processing,
even in the presence of RNA and ssDNA,
such as primers and probes. DNase,
Double-Strand Specific, Heat-Labile
treatment of samples allows for greater
accuracy in the highly sensitive applications
of RT-qPCR and qPCR because signals
from unintended sources of dsDNA are
eliminated. The DNase is ideal for removal
of genomic DNA in RNA preps because
its low inactivation temperature does
not affect RNA stability and the
reverse transcription reaction can be
performed directly in the same tube.
Removal of dsDNA carryover
contamination in PCR mixes before
template addition works without any
Figure 2. Uracil-DNA Glycosylase (UNG), Cod treatment
specifically degrades dUTP-containing DNA in spiked
PCR reactions and inactivates completely to allow
subsequent synthesis of new dUTP-containing
amplicons. Two PCR reactions were prepared with
normal dTTP-containing DNA template and then
spiked with a dUTP-containing amplicon from
a previous PCR reaction. One PCR reaction was
treated with UNG (10063-740) for 5 minutes at
room temperature, while the other tube was left
untreated. The treated and untreated samples were
then immediately amplified by PCR, with the initial
denaturation cycle serving as the heat inactivation
step for the UNG treated sample. Following
amplification, the PCR products with incorporated
dUTP were analyzed by gel. The amplicons were
stored at room temperature for four days and
then analyzed by gel again. In the UNG treated
sample, only one band was present due to effective
degradation of the dUTP DNA. The dUTP DNA
synthesized from dTTP template after UNG heat
inactivation remained stable, even when stored at
room temperature.
reliance upon dUTP substitution in prior
PCR reactions, such as is required for
UNG-based decontamination.
To demonstrate the activity of DNase,
Double-Strand Specific, Heat-Labile, 50ng
of genomic DNA was added to a PCR
master mix that was then divided into two
separate reactions, with one untreated and
the other treated with DNase. Only the
DNase-treated reaction amplified a
detectable signal using an intron-specific
primer in qPCR (Figure 3A, p. 18). DNase
treatment was also performed on an RNA
sample, which was analyzed using the
Eukaryote Total RNA StdSens Assay in
April 2015 I VWRbioMarke Issue 1 I VWR International
17
Figure 3A
Figure 3B
Figure 3. Decontamination of a PCR master mix using DNase, Double-Strand Specific,
Heat-Labile. A PCR master mix containing 50ng genomic DNA was prepared and
divided into two reactions, of which one was treated with DNase, Double-Strand
Specific, Heat-Labile prior to qPCR of both samples. In contrast to the untreated
sample, the DNase-treated sample contained no amplifiable genomic DNA.
DNase, Double-Strand Specific, Heat-Labile treatment leaves RNA quality intact.
RNA incubated with buffer, water or DNase were analyzed using the Eukaryote
Total RNA StdSens Assay (Bio-Rad Experion System), with the results indicating all
samples had intact, high quality RNA (RQI > 8.5).
parallel with control RNA samples. The
data show the quality and quantity of
RNA remain intact after genomic DNA
removal (Figure 3B). This was further
demonstrated through RT-qPCR
experiments comparing target
amplification from cDNA synthesized from
treated and untreated RNA (not shown).
SAN
During protein purification, high salt
concentrations may be used to dissociate
DNA-protein complexes, thereby
improving protein solubility and increasing
accessibility of the DNA to nuclease
activity. High salt conditions, however, are
detrimental to most nucleases. An
exception is VWR Life Science AMRESCO’s
Salt Active Nuclease (SAN), which is
optimized to work at low temperatures in
moderate to high-salt buffers. Its
nonspecific endonuclease activity
efficiently cleaves double- and
18
single-stranded DNA and RNA into mostly
5’-nucleotide oligos. SAN treatment of cell
lysate also reduces sample viscosity, which
is problematic for downstream filtration
and centrifugation steps in the protein
purification process. Removal of SAN
activity following treatment is
achieved by a combination of heating and
addition of a reducing agent, such as DTT.
SAN may also be physically removed by ion
exchange chromatography.
Upon brief centrifugation, only the SAN
treated lysate could form a pellet of debris,
because the untreated lysate was too
viscous. At both temperatures, SAN
degraded DNA into smaller fragments with
increasing units of SAN.
Figure 4. Nuclease activity of SAN in high salt lysis buffer
at both low and high temperatures. DNA removal in E.
coli protein lysates was monitored by agarose gel
electrophoresis following a 30 minute incubation
with increasing units of SAN at 2°C or 37°C.
Description
Size
Cod Uracil-DNA Glycosylase (UNG)
0.1KU
10063-740
Cod Uracil-DNA Glycosylase (UNG)
1KU
10063-742
Heat-Labile Double Stranded DNase (HL-dsDNase)
250U
10147-174
Heat-Labile Double Stranded DNase (HL-dsDNase)
1000U
10147-168
Salt Active Nuclease (SAN)
5KU
10147-170
Salt Active Nuclease (SAN)
25KU
10147-172
Shrimp Alkaline Phosphatase, Recombinant (rSAP)
1KU
10063-736
Shrimp Alkaline Phosphatase, Recombinant (rSAP)
5KU
10063-738
VWR International I VWRbioMarke Issue 1 I April 2015
Cat. No.
Genomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Superior cDNA Synthesis
qScript™ XLT SuperMix
qScript XLT cDNA SuperMix is a next
generation tool for first-strand cDNA synthesis,
providing a highly sensitive and easy to use
solution for two-step RT-PCR and RT-qPCR.
qScript XLT is an engineered M-MLV reverse
transciptase with reduced RNase H activity and
improved activity and stability at higher
A
temperatures. Combined with a precise mixture
of reaction components, this SuperMix enables
superior results over a wide dynamic range of
input RNA, with detectable synthesis of up to
8-fold higher sensitivity than cDNA
synthesis kits using an RNase H(+)
reverse transcriptase (RT).
B
Features
and Benefits
• Improved performance
over traditional reverse
transciptases
• Broad linear dynamic
range
• RNase H minus RT =
higher yield = higher
sensitivity
• Ability to reverse
transcribe difficult
sequences resulting in
improved representation
of problematic sequences
and longer first-strand
product
Figures A and B. Two-step RT-qPCR with high reproducibility, sensitivity, and broad dynamic range. First strand cDNA was
synthesized using qScript XLT cDNA SuperMix from varying amounts of HeLa cell total RNA (1μg to 1pg). Following cDNA
synthesis, 5μL of diluted cDNA product was used as template for qPCR using PerfeCta® qPCR ToughMix® with 0.5X Human
B2M (FAM/MGB) TaqMan® Endogenous Control Assay (Life Technologies).
C
• Out performs ALL first
strand kits on the market
Figure C. Comparison of cDNA synthesis from brain tissue using qScript cDNA SuperMix (, Quanta BioSciences), Competitor
A Master Mix (), and Competitor B Supermix for RT-qPCR (). Using a control kit as baseline, the “-ΔCq” (change in cycle
threshold) is shown for each of 96 genes. qScript XLT cDNA SuperMix shows a 1–2 cycle improvement over Competitor A
MasterMix and 3–4 cycle improvement over Competitor B Supermix, translating into improved sensitivity and yield.
Size
Cat. No.
25 µL x 20
10142-784
100 µL x 20
10142-786
500 µL x 20
10142-788
April 2015 I VWRbioMarke Issue 1 I VWR International
19
Rapid, High-Performance, and
Cost-Effective Plant DNA Extractions
The Challenge of Plant
DNA Extraction
The incredible diversity of the plant
kingdom comes with a wide range of DNA
extraction challenges. These challenges
include extensive variations in plant
structures and in their chemical
composition of polysaccharides,
polyphenolic compounds, and humic
substances, all of which can interfere with
extraction efficiency and/or inhibit
downstream applications.
Omega Bio-tek has long appreciated the
challenges unique to plants and has
worked with many agricultural customers
to develop extraction chemistries,
protocols, and multi-sized kits to optimize
specific plant research workflows.
Because no single extraction method
suits all, Omega Bio-tek has developed a
variety of lysis, washing, and DNA
binding buffers to work with our
assortment of magnetic bead and silica
column chemistries to address plant
extraction challenges.
Performance Analysis of
Extraction Technologies
To demonstrate Omega Bio-tek’s plant
extraction capabilities and to assist
customer selection of the proper
extraction chemistry for their plant of
interest, equivalent starting amounts of
leaf material from 23 of the top agricultural
and biofuel crops were subjected to
automated and manual extraction
performance testing with four different
kits. The automated extractions were
performed using two of Omega Biotek’s
automated Mag-Bind® plant DNA
extraction kit chemistries ported onto a
programmable, open robotic liquid
handler. The manual extractions were
performed with Omega Bio-tek’s E.Z.N.A.®
spin column-based plant extraction kit
along side a similar product from a
leading competitor.
20
Company Q
Automated Method
10128-100
10128-102
Alfalfa
58.4
Apple
Manual Method
Plant DNA DS
101319-368
101319-280
-
85.2
8.9
17.9
10.8
121.8
9.4
5.7
Barley
106.0
198.1
24.9
9.6
Grapes
19.6
212.4
3.9
1.9
Hay
97.8
104.6
41.5
27.2
Oats
157.4
270.0
9.2
18.4
Orange
37.7
31.2
11.9
4.6
Peanut
64.3
52.9
14.6
6.3
Pepper
127.8
111.0
21.1
6.9
Potato
102.1
206.5
24.5
30.0
Sorghum
48.2
72.1
15.7
29.4
Soybean
26.9
25.4
10.5
26.8
Sugar beet
21.0
34.0
2.0
20.2
Tobacco
20.4
19.4
7.5
12.3
Tomato
84.9
120.2
11.1
2.6
Wheat
112.6
152.3
7.0
0.5
Corn
29.6
29.8
5.2
4.0
Cotton
45.8
63.5
13.3
30.5
Sugarcane
121.0
93.1
4.0
10.5
Sunflower
52.8
89.1
15.7
41.8
Canola
34.7
59.0
4.0
3.4
Jatropha
19.6
19.0
14.4
7.5
Switchgrass
35.5
7.9
12.1
21.9
Plant Type
Table 1. Amounts of purified DNA extracted using four different extraction kits. Higher performance in
DNA yields highlighted in purple.
qPCR for β-tubulin gene
qPCR for β-tubulin gene
(0.07)
(0.21)
(1.1)
(1.3)
Pepper Plant DNA (ng)
Figure 1. Bar graph representing the average
real-time PCR cycle threshold value from three
separate real-time PCR reactions of a 0.01 dilution
of the pepper plant leaf extracts obtained from each
of four different extraction methods. The measured
quantity (ng) of DNA in each extract is shown in
parentheses above the associated column. A notemplate control (NTC) reaction is included.
VWR International I VWRbioMarke Issue 1 I April 2015
10128-100
Plant DNA DS
101319-368
Company Q
NTC
10128-100
Plant DNA DS
101319-368
Company Q
NTC
Cotton Plant DNA (1 wng)
Figure 2. Bar graph representing the average realtime PCR cycle threshold value from three separate
real-time PCR reactions of 1.0ng of DNA from the
cotton plant leaf extracts as obtained from four
different extraction methods. A control no-template
control (NTC) reaction is included.
Genomics
The results in Table 1 reveal that the
automated methods resulted in significantly
higher recoveries of DNA than either of the
manual methods tested. Of course, the
automated methods were also much faster
and involved much less hands-on time than
the manual methods. Interestingly,
depending on plant type, maximum DNA
yields were obtained by either of the two
different Omega Bio-tek automated
extraction technologies. For the manual
extraction methods, our more cost-effective
product performed on par with the leading
competitor product. Altogether, these
results demonstrate the benefit of Omega
Bio-tek’s multiple technical approaches for
plant DNA extraction.
Because the extraction methods being
tested are based on different chemistries,
it is possible that differences in the
chemical composition of the resulting
extracts might adversely influence DNA
quantification, otherwise known as “matrix
effects”. To ensure that the DNA quantity
measurements were accurate, real-time
PCR for the β-tubulin gene was performed
on a subset of 100-fold dilutions of plant
extracts from all four methods. The
differences in the resulting real-time PCR
cycle threshold values were consistent with
the amount of template measured to be
present in each extract (Figure 1), or when
adjusted to contain equivalent (1ng)
amounts of template (Figure 2). These
results indicate that matrix effects have not
substantially influenced reported DNA
recovery results.
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
1500rpm. DNA was extracted using
Omega Bio-tek’s automated technologies
(Cat. No. 10128-100, Mag-Bind® Plant DNA
Plus 96 kit; and Mag-Bind® Plant DS 96
DNA kit) ported onto an open and
programmable automated liquid handler
(Qiagen BioSprint 96). Manual extractions
were performed using Omega Bio-tek spin
column technology (Cat. No. 101319-280)
and a kit from a leading competitor
(company Q), following manufacturer’s
instructions. Resulting purified DNA was
quantified via Promega’s QuantiFluor® DS
DNA system and normalized per mg of
plant material input. Real-time PCR was
performed with Agilent’s Brilliant III 2X
SYBR® mix and primers specific for the
β-tubulin gene using a standard
amplification protocol on the ABI 7900.
Conclusion
Omega Bio-tek has leveraged their
plant extraction experience, and
expertise to develop a robust,
customizable, and multi-tiered
solutions for plant DNA extraction needs.
Options are available for automation on
open liquid handling and magnetic
processor platforms such as the
Hamilton Microlab STAR and Thermo
Scientific™Kingfisher™ Flex.
Description
Preps
E.Z.N.A. SP Plant DNA Kit
50
101319-368
200
101319-280
Mag-Bind Plant
DNA Plus Kit
Cat. No.
1 x 96
10128-100
4 x 96
10128-102
Methods
Plant specimens were acquired
commercially or grown at Omega Bio-tek.
Each extraction of each plant type was
performed in triplicate for each method
tested. Each sample consisted of
approximately 50mg (wet weight) of
plant leaf material and was disrupted at
April 2015 I VWRbioMarke Issue 1 I VWR International
21
Robust and Highly-Specific Multiplex PCR
Using Q5® High-Fidelity DNA Polymerase
Julie F. Menin, M.S. and Nicole M. Nichols, Ph.D.
New England Biolabs, Inc.
Introduction
Multiplex PCR is a type of polymerase
chain reaction (PCR) in which numerous
pairs of primers are used to amplify
multiple targets in a single experiment.
This technique is routinely used in
genotyping, pathogen detection, and
enrichment techniques. Although primer
design is arguably the most significant
contributing factor to multiplex PCR
success, reactions employing Taq-based
enzymes can also require significant
optimization. Among those components
that most often require optimization are
Mg++, dNTPs, primer, and enzyme
concentrations. Specific mixes that provide
optimized compositions for Taq-based
multiplexing reactions are commercially
available, but for some users, maintaining
a mix just for multiplexing needs is not
convenient. In addition, for PCR-based
DNA enrichment upstream of next
generation sequencing, the lower fidelity
of Taq-based mixes can be problematic.
For this study, we sought to determine
how our ultra-high fidelity Q5 DNA
Polymerase products performed in
multiplex PCR.
Q5 High-Fidelity DNA Polymerase is
composed of a novel polymerase that
is fused to the processivityenhancing Sso7d DNA binding
domain, improving speed, fidelity,
and reliability of performance. In
work described elsewhere,
Q5’s fidelity has been
determined by both
traditional blue-white assay
methods and Sanger
sequencing to be at least
100X higher than that
of Taq DNA
Polymerase.
22
Table 1: Target Details
Amplicon
Size
(bp)
GC Content
Ta**
A
723
59%
72°C
B
547
40%
68°C
C
331
36%
66°C
D
139
52%
72°C
* Unless otherwise noted, materials were obtained
from, and manufactured by, New England
Biolabs® (NEB®), Ipswich, MA.
** Annealing temperature (Ta) as calculated by the
NEB Tm Calculator for experiments using Q5.
Materials & Methods*
Experiments shown in this application note
employed Q5 High-Fidelity 2X Master Mix
and primer sets designed to amplify four
targets from human genomic DNA
(Xp21.2, 19p13.2, and two from Xp21.1).
These targets ranged from 36-59% GC
content (Table 1) and could easily be
resolved using traditional agarose-based
gel methods or a higher-throughput
microfluidic-based system. Primer sets
were designed according to
recommendations provided with the
product and were previously
demonstrated to be appropriate for
multiplex experiments using Taq DNA
Polymerase. Except where otherwise
noted, Q5 DNA Polymerase products were
used according to the manufacturer’s
recommendations.
Reaction Setup
Because Q5 typically requires higher
annealing temperatures than Taq-based
products, the NEB Tm Calculator was used
to determine appropriate annealing
temperatures for each primer set.
Amplification reactions were either set up
on ice and added to a pre-heated
thermocycler or, for hot start enzymes, set
up at room temperature and added to a
room-temperature thermocycler.
VWR International I VWRbioMarke Issue 1 I April 2015
Amplification results were visualized
either by traditional ethidium bromide
staining and agarose gel electrophoresis or
via a microfluidic-based LabChip® GXII
system using a 5K DNA/RNA chip. For
analysis on the LabChip, samples were first
diluted 1:1 with water to reduce mobility
differences that can arise from buffer
effects. Primary data (electropherograms)
are converted to virtual gels by the
machine software. Settings for all
experiments used either default software
values (v.4.0.1418.0) or, when modified,
used the same modifications across all
lanes to permit comparisons within and
between experiments.
Results
To determine an annealing temperature for
subsequent experiments, a gradient PCR
with annealing temperatures from 52-72°C
was conducted [98°C/30s, 30x(98°C/10s,
52-72°C/30s, 72°C/30s), 72°C/5m] using Q5
High-Fidelity 2X Master Mix. As seen in
Figure 1, annealing temperatures from
60-67°C supported specific and robust
amplification of all four targets, without any
additional optimization of reaction
components. For remaining experiments,
an annealing temperature of 65°C was
used. Reaction components that often
require optimization for multiplex
experiments using Taq DNA Polymerase
were then modified to determine whether
Q5 would also benefit from similar
optimizations. As seen in Figure 2, none of
the methods for optimization investigated
(increasing concentrations of dNTPs,
enzyme, primers or Mg++) offered significant
improvement over standard Q5 reaction
conditions. In addition, for these four
targets, increasing the Mg++ concentration
over 3mM had a deleterious effect on yield,
though specificity remained high.
Genomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Figure 1. Effect of annealing temperature (Ta) on Q5 multiplex PCR. Q5 HighFidelity 2X Master Mix was used in a gradient PCR (Ta from 52-72°C) to
determine conditions that would support robust and specific amplification
of four human gDNA targets (A-D). Reactions were diluted 1:1 with water
before being visualized on a LabChip GX II system.
Figure 2. Q5 multiplex PCR optimization. Using an annealing temperature
of 65°C, reaction components that are commonly optimized to ensure
robust multiplex PCR results were varied. The final concentration of the
component varied in each experiment is indicated above each panel.
For each experiment, the first non-ladder lane represents the typical
recommended conditions for Q5. Few modifications were able to
improve amplification of the four human gDNA targets examined (A-D)
compared to standard Q5 conditions, which supported specific and robust
amplification. Mg++ concentrations greater than 3mM were inhibitory.
Experiments conducted to examine the
lower limits of primer concentration
showed robust yields and high specificity
using concentrations as low as 0.13μM for
each primer (data not shown).
Further investigation of the Mg++ -related
reduction in yield using 2X enzyme (0.04
U/µL in the final reaction, achieved by the
addition of stand-alone Q5 into the
master mix) demonstrated that Mg++ related inhibition was not relieved by
increasing the polymerase concentration
(data not shown). Interestingly, specificity
was maintained over all reaction
conditions investigated.
Conclusion
Using Q5 High Fidelity DNA Polymerase
2X Master Mix, we demonstrated
successful multiplex amplification of
human targets using a wide range of
annealing temperatures, from 0.13 to
2μM primers, from 0.02–0.06 U/μL and
up to 4mM Mg++. These studies and
additional work investigating larger
primer sets (n ≥12) and other commercially
available forms of Q5 (data not shown)
Description
suggest that
Q5 DNA Polymerase
is capable of robust and
highly specific multiplex PCR
results with little-to-no
optimization, other than typical
primer design criteria. Q5 offers not
only ultra high-fidelity amplification
critical for downstream workflows, but
also a convenient option for
multiplex PCR.
Cat. No.
Q5 High-Fidelity DNA Polymerase
102500-138
Q5 High-Fidelity 2X Master Mix
102500-134
Q5 Hot Start High-Fidelity DNA Polymerase
102500-146
Q5 Hot Start High-Fidelity 2X Master Mix
102500-142
Deoxynucleotide (dNTP) Solution Mix
101228-418
April 2015 I VWRbioMarke Issue 1 I VWR International
23
FlashGel™ System for DNA Recovery
Mary Riley and Hugh White, Lonza Rockland, Inc.
System enables recovery of DNA samples
directly from the gel, in a simple procedure
that takes just 4–10 minutes, depending
upon sample type. The system eliminates
the need to cut away and then purify
bands, maximizes the efficiency of
recovered DNA, and minimizes the amount
of handling post-recovery.
Introduction
Direct DNA recovery using the FlashGel
Recovery System eliminates agarose gel
preparation, band excision, and purification,
and delivers highly efficient recovery, free
from inhibitors and UV-induced damage, in
a simple 5–10 minute protocol.
DNA recovery post-agarose gel separation
is a fundamental tool of molecular biology
research. Basic techniques for band
excision and spin column DNA purification
have evolved very little in the past several
decades and present several areas of
concern for the researcher. First, band
excision requires careful removal of
agarose material to avoid loss of DNA and
minimize agarose in the sample. This
process requires DNA damaging UV light
to visualize the DNA bands to be removed.
Second, column purification requires
careful and precise placement of elution
buffer in the membrane, monitoring of pH
sensitive binding and elution, and user
caution in carrying over residual ethanol
from wash steps. Finally, a minimum of one
24
hour is required for the entire process of
separating a DNA sample on an agarose
gel, excising it from the gel, and purifying it
on a spin column.
We have developed the FlashGel Recovery
System, which eliminates both the
preparation and wait time associated with
gel electrophoresis, and the band excision
and purification steps associated with
recovery. The entire process is reduced to
10 minutes or less, with greater than 80%
recovery efficiencies. The recovered sample
is free of inhibitors and UV-induced
mutations, and subsequent reamplification, cloning and ligation is
equivalent or superior to columnrecovered DNA.
Product Overview
The FlashGel System revolutionized DNA
separation by combining electrophoresis
speed and visible light illumination, such
that separation is completed in just 5
minutes and band migration can be
viewed in real-time. The FlashGel Recovery
VWR International I VWRbioMarke Issue 1 I April 2015
As DNA migrates to the second tier of wells,
it is free from the agarose matrix and easily
extracted via pipette, with the aid of the
FlashGel Recovery Buffer. Visible light from
the compact FlashGel Dock illuminates the
recovery wells without damage to the DNA
or hazard to the user. Samples are recovered
at 80–100% efficiency, are free of inhibitors,
and ready for subsequent re-amplification,
cloning, or other techniques. The
proprietary stain in the FlashGel Cassettes
enables separation and recovery of very
small quantities of DNA, and minimizes user
exposure to potential mutagens.
Depending upon initial separation time in
the recovery step, the same cassette may be
used for analysis verification of the
recovered sample.
Methodology
Several experiments were conducted to
demonstrate performance in typical
preparative applications.
FlashGel Recovery Capabilities
To demonstrate general recovery efficiency,
serial doubling dilutions (18.25–600ng) of
1000bp BioMarkers® Purified DNA
Fragments (BioVentures, Inc.) were
separated and recovered using a FlashGel
Recovery Cassette. Briefly, the samples were
loaded in to the top tier of wells and run to
the top edge of the second tier of wells. The
run was stopped and 20μL FlashGel
Recovery Buffer was added to the
appropriate wells in the second tier. The run
was recommenced and the fragment run
fully into the wells of the second tier. The
DNA was recovered by reverse pipetting
Genomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
from the wells. 5% of each recovered
sample was subsequently analyzed using a
1.2% FlashGel DNA Cassette. Comparison to
the FlashGel QuantLadder run on the gel
showed that recoveries appeared to range
from 80–90%.
DNA Recovery and Cloning
Plasmid DNA (pBr322; New England
BioLabs) was subjected to restriction
enzyme double digestion using PstI and
BamHI (New England BioLabs). Samples of
the restricted DNA were separated and
3.2kb DNA fragments were recovered
using the FlashGel Recovery System (FG) or
a spin column kit (C). 5% of each recovered
DNA sample was analyzed on a 1.2%
FlashGel DNA Cassette (Fig. 1A). Aliquots
of the recovered DNA samples were
ligated into PstI /BamHI double digested
pUC19 vector using the Rapid DNA
Ligation Kit (Fermentas, Inc.) and
transformed into NEB 5-alpha E. coli
Competent Cells (New England BioLabs).
The number of colonies obtained with
both samples were very similar.
Plasmid samples from two colonies
from each sample were digested with
PstI/BamHI and analyzed on a 1.2%
FlashGel DNA Cassette along with a
restricted sample of vector with no insert
(Fig. 1B). The data clearly shows appropriate
excision of the expected insert.
PCR Amplification of
Recovered DNA
Reagents from the GeneAmp® Gold Kit
(Applied Biosystems) were used to amplify
300 and 500bp fragments in a multiplex
PCR amplification reaction. A 6μL aliquot
of the PCR reaction was separated on the
FlashGel Recovery System and the PCR
product bands were recovered. 4μL of
both recovered PCR fragments were
analyzed on a 1.2% FlashGel DNA Cassette
(Fig. 2A). Aliquots of the recovered
fragments were also used as templates for
Panel A –­ Recovered Samples
Panel B –­ Plasmid Restriction Digests
FGC
FG1 FG2C1 C2 V
Figure 1: Recovery and cloning comparison. Samples of PstI/BamHI cut pBr322
were separated and 3.2kb DNA fragments were recovered using the FlashGel
Recovery System (FG) or spin column kits (C1 and C2). (A) shows the comparison
between 5% of each recovered DNA sample separated on a 1.2% FlashGel DNA
Cassette, along with the FlashGel QuantLadder. (B) PstI/BamHI cut plasmid
samples from colonies transformed with recovered DNA samples; lane “V” is
digest of vector with no insert. Other lanes contain the FlashGel QuantLadder and
FlashGel DNA Marker (100bp to 4kb).
Panel A –­ Recovered PCR
Products
new PCR amplifications. Fig. 2B shows
0.5μL aliquots of these amplification
reactions separated on a FlashGel DNA
Cassette. The results show that the
recovered DNA could be used successfully
as a template for amplification.
Summary
The FlashGel Recovery System is a fast and
effective tool for most preparative
applications, providing users with an
alternative method that both maximizes
recovery efficiency and minimizes
opportunity for damage to precious DNA.
When used as a combined preparative and
analytical tool, the FlashGel Recovery
System is a highly economical method to
transform a laboratory DNA workflow.
Description
Cat. No.
FlashGel Recovery System
95045-604
FlashGel Recovery Starter Kit
95053-314
FlashGel Recovery Kit
89400-690
FlashGel Recovery Cassette
89135-718
Panel B –­ Plasmid Restriction Digests
1 234
Figure 2: PCR amplification of recovered DNA. Reagents from the GeneAmp Gold
Kit were used to amplify 300 and 500bp fragments in a multiplex PCR amplification
reaction. 6μL of the PCR reaction was separated on the FlashGel Recovery System and
the PCR product bands were recovered. (A) 4μL of both recovered PCR fragments.
The recovered fragments were used as templates for new PCR amplifications. (B)
0.5μL aliquots of these amplification reactions. The resulting amplification products
were observed for all reactions by gel analysis on a 1.2% FlashGel DNA Cassette,
along with the FlashGel QuantLadder. Reactions 1 and 3 used primers for 300 and
500bp fragments while reactions 2 and 4 used only single primer set.
April 2015 I VWRbioMarke Issue 1 I VWR International
25
qTOWER 2.0/2.2:
Set New Standards in
Real-time Quantitative PCR
Featuring a striking, modern design, qTOWER
2.0 and 2.2 allow quantitative PCR in an
established 96-well SBS standard format. These
systems offer an open platform for many
types of real-time PCR plastic materials, such
as 0.2mL single tubes, 8-well strips, or
96-well microplates.
The high-quality gold plated silver block
ensures an outstanding level of temperature
homogeneity of 0.2°C along the entire block
and is therefore ideally suited for all real-time
PCR applications.
In combination with the optional gradient
function (qTOWER 2.2 model), different assays
can be optimized with minimum effort. The
qTOWER 2.0 and 2.2 are equipped with a
patented, fiber-optic shuttle system for the best
possible excitation and detection of a variety of
known fluorescence dyes, eliminating the need
for cumbersome passive reference protocols.
oNE dECISION - mANY advantages
•
Quantitative real-time PCR in a proven 96-well SBS standard format
•
A 10-year warranty on all system optical components
•
Ramping rates of up to 5.5°C/sec.
•
Optimal for volumes of 10–60 μL
•
Linear Gradient Tool (LGT) with max. temperature
gradient span of 40°C
•
Ideal detection homogeneity, with a patented fiber optic
shuttle system
•
Individual configuration with up to six different measurement channels
•
Choose from 12 high-resolution, retrofittable FRET, color, and
protein modules
•
License-free control and analysis software, including a wide
variety of easy-to-use results analysis methods
Patented Fiber Optic Shuttle System
The qTOWER 2.0/2.2 works with three independent, long-life
LEDs to optimally excite all applicable fluorescent dyes. Thus, the
system can process sophisticated multiplex experiments with up
to six different fluorescently-labeled probes ranging from blue to
the far-red spectral range. Moreover, the patented optical system
consists of a shuttle with eight high performance fibers, which
guarantee a homogenous read-out of the 96-well block within
6s, regardless of the number of dyes to be measured.
Each component of the high performance fiber
optical system has a 10-year warranty!
26
VWR International I VWRbioMarke Issue 1 I April 2015
Genomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
qPCRsoft – Clear, Structured
Control and Analysis Software
for Every Need
A. Amplification plots linear view
B. Amplification plot logarithmic view
Figure 1. Linear amplification over 9 logs by using qTOWER 2.2 and a dilution series of yeast genomic DNA from
109 to 101 copies. The PCR efficiency of > 0.98 with R² = 0.9994 for amplifying a yeast specific target sequence
was determined automatically by qPCRsoft
A. Amplification plots linear view
B. Amplification plot logarithmic view
Figure 2. Amplification of an E.coli specific target sequence in 96-well format using qTOWER 2. The analysis of
Ct values with a mean Ct of 12.99 and a standard deviation of 0.08 shows the excellent homogeneity of the
thermal silver block and high performance optics.
The qPCRsoft control and analysis
software offers the highest level of
flexibility and ease of use. The logical
arrangement of all tools, intuitive user
interface, parameter-orientated
memory and programming concept
provide a major advantage in using the
qTOWER 2.0/2.2. While a cycle is in
process, the operator can easily
evaluate the data of previous
experiments in parallel.
qPCRsoft Features and
Benefits Include:
• Integrated evaluation algorithms,
including absolute and relative
quantification, delta-delta
Ct method, PCR efficiency, allelic
discrimination, endpoint analysis
• Also applicable for protein
thermal shift assays
• Parameter-orientated program
guides
• User management with three
authorization levels
• MIQE compliance
Description
Cat. No.
qTOWER 2.0, No Gradient
10066-140
qTOWER 2.2, With Gradient
10066-144
April 2015 I VWRbioMarke Issue 1 I VWR International
27
Using the DuPont™ BAX® System to Detect
Salmonella, E. coli O157:H7 and Non-O157
STEC From a Single Beef Enrichment
For more information on
the DuPont BAX System,
please contact your
VWR representative.
Introduction
To demonstrate the convenience and
robustness of the DuPont BAX System, DuPont
Nutrition & Health evaluated a single
enrichment medium for testing beef samples
for Salmonella, E. coli O157:H7 and the top six
non-O157 Shiga toxin-producing E. coli (STEC).
In this study, 325g samples of artificially spiked
beef trim, ground beef, and lean finely textured
beef were tested with the BAX System method
after enrichment in modified TSB with
casamino acids and 8mg/L novobiocin
(mTSB+n). Results of the alternative test
method were compared to the appropriate
USDA-FSIS reference method as published in
the Microbiology Laboratory Guidebook.
The results of this study demonstrate that the
BAX System can effectively detect Salmonella,
E. coli O157:H7, stx/eae and the top six nonO157:H7 STEC from 325g samples of beef trim,
ground beef, and lean finely textured beef
after enrichment in mTSB+n.*
* Note: STEC testing for lean finely textured beef
was not included in this study.
was streaked for purity on BHI agar, then
suspended in BHI broth and incubated at 37°C
overnight to an assumed concentration of 109
cfu/mL. Strains were then serially diluted in
additional BHI broth to levels likely to produce
fractional positive results based on internal
preparatory studies.
Samples of beef trim (15% fat), ground beef
(15% fat, 20% soy protein), and lean finely
textured beef were provided by qualified beef
producers and pre-screened for the targets of
interest. For each sample matrix and test strain
combination, 25g portions were removed from
a master sample and spiked with the
appropriate strain dilution at levels expected to
yield a concentration of approximately 1 cfu/
portion. Five 25g portions of each sample
matrix were removed and left unspiked to
serve as negative controls. Spiked samples were
re-mixed to create a spiked master sample and
held at 4°C for two days in order to stress the
target organism, then divided into 25g
portions for enrichment and testing.
Sample Enrichment
Methods
Strain Selection and Preparation
A variety of Salmonella and E. coli strains
originally isolated from cattle were selected
from the DuPont Nutrition & Health Culture
Collection for use in this evaluation. Each strain
28
VWR International I VWRbioMarke Issue 1 I April 2015
For the BAX System method, each 25g portion
was combined with 300g unspiked matrix to
create 325g samples for testing. Each 325g
sample was then added to 975mL pre-warmed
(35°C) modified TSB with casamino acids and
8mg/L novobiocin (mTSB+n), stomached or
hand-massaged for 2 minutes and incubated at
Genomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
this study are available from DuPont Food
Diagnostics upon request.
42°C, with aliquots removed at 15 and
22-24 hours for testing with the BAX
System method.
For the USDA-FSIS reference method,
samples were prepared and enriched for
each sample matrix according to the
method published in the Microbiology
Laboratory Guidebook at the time of
the evaluation.
BAX System Method
For E. coli O157:H7 and STEC testing,
20μL enrichment was added to 200μL
prepared BAX System lysis reagent in cluster
tubes. For Salmonella testing, 5μL
enrichment was added to lysis reagent in
cluster tubes. For all samples, lysis was
performed by heating tubes at 37°C for 20
minutes and 95°C for 10 minutes, then
cooling tubes at 4°C for at least 5 minutes.
For E. coli O157:H7, STEC and real-time
Salmonella testing, 30μL of each lysate was
used to hydrate a PCR tablet for the
appropriate BAX System assay. For the
Salmonella and Salmonella 2 tests, 50μL of
lysate was used. PCR tubes were loaded into
the BAX System Q7 instrument and a full
process was run according to the procedure
described in the BAX System User Guide.
All samples, regardless of BAX System
result, were confirmed according to the
reference culture method as described in
the USDA-FSIS Microbiology Laboratory
Guidebook.
For Salmonella testing, Pearson’s Chi Square
analysis for unpaired samples demonstrates
that there is no statistically significant
difference between the results of the test
methods and the corresponding reference
method (X2 < 2.7055). For E. coli O157:H7
testing, McNemar’s Chi Square analysis for
paired samples demonstrates that there is
no statistically significant difference
between the results of the test methods
and the corresponding reference method
(X2 < 3.84) for all sample types except
ground beef tested after 12 hours
enrichment. For non-O157 STEC testing,
McNemar’s Chi Square analysis
demonstrates that there is no statistically
significant difference between the results of
the test methods and the corresponding
reference method (X2 < 3.84).
Conclusions
Results
A summary of the comparison between
results of the BAX System method and
results of the USDA-FSIS reference method
is displayed in the table. The full results of
The results of this study demonstrate that
the BAX System can effectively detect
Salmonella, E. coli O157:H7, stx/eae and the
top six non-O157:H7 STEC from 325g
samples of beef trim, ground beef, and lean
finely textured beef from a single
enrichment in mTSB+n.
BAX System Method vs. USDA FSIS Method Results
Real-time
Salmonella
15 hr
Beef
trim
Ground
beef
Textured
beef
Salmonella
24 hr
15 hr
24 hr
Salmonella 2
Real-time E. coli
O157:H7
STEC Screening
STEC Panel 1
STEC Panel 2
15 hr
15 hr
15 hr
15 hr
15 hr
24 hr
24 hr
24 hr
24 hr
24 hr
Test Pos*
7/10
7/10
7/10
7/10
7/10
7/10
5/10
5/10
6/10
6/10
0/10
0/10
6/10
6/10
FSIS Pos
9/10
9/10
9/10
9/10
9/10
9/10
5/10
5/10
6/10
6/10
0/10
0/10
6/10
6/10
Chi Square
1.1
1.1
1.1
1.1
1.1
1.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Test Pos*
36/60
39/60
37/60
39/60
37/60
39/60
22/60
22/60
19/30
19/30
19/30
19/30
0/10
0/10
FSIS Pos
36/60
36/60
36/60
36/60
36/60
36/60
22/60
22/60
19/30
19/30
19/30
19/30
0/10
0/10
Chi Square
0.0
0.28
0.04
0.28
0.04
0.28
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Test Pos*
7/10
7/10
7/10
7/10
7/10
7/10
7/10
7/10
--
--
--
--
--
--
FSIS Pos
6/10
6/10
6/10
6/10
6/10
6/10
7/10
7/10
--
--
--
--
--
--
Chi Square
0.22
0.22
0.22
0.22
0.22
0.22
0.0
0.0
--
--
--
--
--
--
*Indicates total number of presumptive positive results reported by the BAX System software
April 2015 I VWRbioMarke Issue 1 I VWR International
29
OPTIMIZER PCR Workstation™
Improve Accuracy of Sensitive PCR Amplification Reactions
The OPTIMIZER PCR Workstation is
designed to provide an optimal
environment for performing PCR
amplification reactions.
The Workstation interior with
solutions, reagents, and equipment
inside may be irradiated prior to use.
Exposure to powerful UV blocks
replication of potentially contaminating
DNA sequences by causing adjacent
pyrimidines to undergo dimerization1,2.
The PCR Workstation is equipped with two
(dual) ultraviolet lamps that emit light with
a 254nm wavelength. When solubilized
DNA is exposed to this radiation, adjacent
thymine bases will be induced to form
cyclobutane pyrimidine dimers by the
condensation of two ethylene groups at
C-5 and C-6.
Additionally, adjacent thymines can be
linked between the C-4 residue and
the C-6 of its neighbor. In either case, a
“kink” is introduced into the DNA.
Therefore, by pre-exposing the PCR
work area with UV radiation, all DNA
present will be photo-damaged and
will not be amplified by DNA
polymerase (despite retaining the
ability to be primed). Consequently,
these photo-damaged sequences will
not contaminate your PCR
amplification product3.
The protected area within the Workstation
also limits exposure of the experimental
set-up to the open lab environment,
decreasing the chances of cross or
airborne contamination.
Ergonomically designed for comfortable
posture, OPTIMIZER PCR Workstations
are available in six different models. The
size options available are 24 inches deep x
24 inches high, with widths of 30, 36, and
30
VWR International I VWRbioMarke Issue 1 I April 2015
48 inches. Each size is equipped with dual
UV lights and dual fluorescent lights built
into the ceiling.
Work surface can be stainless steel or
chemical resistant black Formica. For
added convenience, a twelve-hour
countdown timer controls the UV
irradiation dosage, and can be set
to a pre-determined time
for decontamination.
The PCR Workstation can be placed on a
lab bench, or turned into a moveable work
area by ordering an accessory cart with
locking casters.
Dual UV bulbs help irradiate areas that
might otherwise be inaccessible with a
single bulb. The dual UV bulb format is
required when the researcher desires to
use the Workstation to decontaminate
apparatus and reagents.
Aerosols of previously amplified DNA
contaminants can often be found on
racks, pipettes, tubes, or reagent bottles.
These potential sources of contamination
need to be irradiated but when placed in a
PCR workstation can create “shadow”
areas where the UV dosage from a
single bulb can be insufficient for
decontamination. To help solve this
problem, the two UV bulbs are mounted
apart from each other in the ceiling,
maximizing the contents of the
Workstation that will receive direct
UV irradiation.
The stainless steel ceiling angle reflector
and reflective white sides of the PCR
Workstation provide enough UV radiation
bounce to get to all shadowed areas. The
dual bulb format also shortens the amount
of exposure time necessary. Dual bulbs
may also be required to deliver the
sufficient UV dosage needed to prevent
Genomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Each OPTIMIZER PCR Workstation is equipped with the following:
•
Chemical-resistant black Formica or Stainless Steel work surface
•
Tempered safety glass screen
•
Duplex electrical outlet mounted in ceiling
•
Two fluorescent lamps mounted in ceiling assuring
excellent work space visibility
•
A stainless steel ceiling is used for greater UV
reflection, resulting in a 20% increase with
dual UV bulbs.
•
Dual 254nm UV Germicidal lamps with 12 hour
countdown timer with time-hold position
•
Single or double doors can be closed to prevent
contamination, and can be conveniently stowed
away during experimental procedures by sliding
into storage compartment in the base of the
PCR Workstation
•
Hinged glass screen allows easy access to interior
workspace for cleaning and placement of
large instruments.
•
Factory installed safety interlock automatically
shuts off UV lights when door is opened.
Intensity of UV Light for Dual UV Bulb OPTIMIZER PCR Workstation
CBP-030-202 or CBP-030-202-SS Dual UV
Bulb Workstation
CBP-036-202 or CBP-036-202-SS Dual UV
Bulb Workstation
CBP-048-202 or CBP-048-202-SS Dual UV
Bulb Workstation
µW/cm@ UV @ worksurface
400
400
800
Exposure time of Aqueous DNA
30 minutes
30 minutes
15 minutes
Exposure time for Dry DNA
2-8 hours
2-8 hours
1-4 hours
replication of certain types of
contaminating DNA. For example,
inactivation of dry DNA requires
more UV exposure than that of DNA
in solution.
To determine the best suitable UV
irradiation dosage for the type and source
of contaminating DNA, please refer to the
table above or references 1, 2, and 4 for the
exposure times.
References
1. Sakar, G., and Sommer, S, (1990) Nature 343 p. 27.
2. Ou, C-Y, Moore, J.L. and Schochetman, G Biotechniques (1991)
10:4 p. 442-445.
3. Lehninger, A.L., Nelson D.L., and Cox, M.M., (1993) Principals of
Biochemistry. Worth Publishers, New York, NY pages, 342, 816,
832, 837.
4. Fairfax, M.R., Metcalf, M.A., Cone, R.W., (1991). PCR Methods and
Applications. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York. 1:142-143.
Width, cm (in.)
Cat. No.
Black Laminate Work Surface
76.2 (30)
CBP-030-202
91.4 (36)
CBP-036-202
121.9 (48)
CBP-048-202
Stainless Steel Work Surface
76.2 (30)
CBP-030-202-SS
91.4 (36)
CBP-036-202-SS
121.9 (48)
CBP-048-202-SS
April 2015 I VWRbioMarke Issue 1 I VWR International
31
Even the Smallest Thing
Can Have a Big Impact
GE Healthcare has decades of experience in chromatography and strives to
continuously provide researchers with tools to deliver better results, higher resolution,
and faster runs. Significant improvements in a chromatography workflow can be
achieved with small changes. With smaller, more rigid agarose beads, we have designed
new chromatography media (resins) that tolerate higher flow rates, which in turn have
a positive impact on the speed and resolution that you can achieve in size exclusion
chromatography experiments. Switching to these new media, Superdex™ 200 Increase
or Superose™ 6 Increase, which are replacing our popular Superdex 200 and
Superose 6 media , respectively, requires minimal changes to your protocols running in
non-regulated environments. When you switch, you will be rewarded with up to 50%
increased resolution, or half the run time with the same resolution as before.
Superdex 200 Increase Columns for
MAb Purification and Analysis
Superdex 200 Increase columns are excellent
for the quantitation of antibody monomers,
dimers, and aggregates in monoclonal
antibody (MAb) preparations. The increase in
resolution for Superdex 200 Increase compared
to Superdex 200 enables detection of
monoclonal antibody fragments (Fig 1).
Superose 6 Increase for Purification and
Analysis of Large Protein Complexes
Purification and analysis of large protein
complexes is an area of intense research
effort. Purification of such complexes is
often a challenge. Superose 6 Increase has
32
VWR International I VWRbioMarke Issue 1 I April 2015
been specifically designed for the separation
of molecules in the molecular weight range
of 5 000 to 5 000 000. Superose 6 Increase
is an excellent choice for use as a
complement to tandem affinity purification
(TAP) for the final purification of protein
complexes and to provide a size
estimate of the purified complex in its
native state.
The run time using Superose 6 Increase
can, in general, be cut in half as compared
to its predecessor Superose 6 (Fig 2).
Proteomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Convenient Prepacked Columns
Both Superdex 200 Increase and Superose
6 Increase are available in three different
sizes of prepacked columns, suitable for
both small scale preparative and analytical
purposes (Fig 3).
Superdex 200 Increase and Superose 6
Increase columns provide:
• Higher purity due to higher resolution
• Shorter run times due
to the higher flow
rates
• Versatile use for both
preparative and
analytical applications
• Tolerance for high pH,
harsh cleaning protocols for
long column life time and minimal
protein carry over
Figure 1
Superdex 200 Increase 10/300 GL
130301 10 300 10136375 Mab5 nr 2 001:10_UV1_280nm
130301 10 300 10136375 Mab5 nr 2 001:10_UV1_280nm
Superdex 200 10/300 GL
130301 10 300 10136375 Mab5 nr 2 001:10_Inject
130306 SDX200 old 10111147 Mab5 001:10_UV1_280nm
130301 10 300 10136375 Mab5 nr 2 001:10_Inject
mAU
130306 SDX200 old 10111147 Mab5 001:10_Inject
mAU
mAU
130306 SDX200 old 10111147 Mab5 001:10_UV1_280nm
130306 SDX200 old 10111147 Mab5 001:10_Inject
mAU
15.0
300
300
Rs 2.7
15.0
Rs 1.7
10.0
250
250
Figure 3. (above) Superdex 200 Increase and Superose
6 Increase are available in three different column
sizes (10/300 GL, 5/150 GL, 3.2/300) to fit different
application needs.
10.0
5.0
5.0
200
200
0.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
ml
0.0
150
150
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
ml
100
100
50
50
0
0.0
0
0.0
10.0
20.0
30.0
40.0
50.0
10.0
20.0
30.0
40.0
50.0
min
min
Figure 1. (above) Superdex 200 Increase delivers up to 50% better resolution for monoclonal antibody
purification (MAb concentration 3.4mg/mL) as compared to Superdex 200 at the same flow rate (0.5mL/min).
Figure 2
1.0 ml/min -> 24 min
0.5 ml/min -> 48 min
Figure 2. Superose 6 Increase
can deliver results twice as
fast, while retaining resolution,
as compared to Superose 6
(sample contained a mix of
standard proteins).
Description
Cat. No.
Superose 6 Increase 10/300 GL
10192-228
Superose 6 Increase 5/150 GL
10192-230
Superose 6 Increase 3.2/300
10192-226
Superdex 200 Increase 10/300 GL
89497-272
Superdex 200 Increase 5/150 GL
89497-274
Superdex 200 Increase 3.2/300
89497-276
Superose 6 Increase 10/300 GL Superose 6 10/300 GL
April 2015 I VWRbioMarke Issue 1 I VWR International
33
A Luminol- and Peroxide-Based
Chemiluminescence System for the Sensitive
Detection of Horseradish Peroxidase (HRP) for
Western Blotting Detection of Proteins
G-Biosciences’ new formulation of picoLUCENT™ PLUS was compared to two competitive highly sensitive reagents
with reported similar sensitivity to G-Biosciences’ picoLUCENT PLUS. picoLUCENT PLUS significantly outperformed
one product and was a slight improvement on the other competitor’s product. In addition to sensitivity,
G-Bioscience’s product was significantly more affordable than both competitors’ products.
INTRODUCTION
with NAP-BLOCKER™ (Cat. No. 82022626). The housekeeper protein actin was
detected with a rabbit polyclonal
antibody (Santa Cruz Biotechnology, Inc.)
at a concentration of 1:5000, as opposed
to the recommended 1:100 to 1:1000. The
primary antibody was detected with a
secondary antibody conjugated to HRP.
Working solutions of G-Biosciences’
picoLUCENT PLUS, Competitor T’s
substrate, and competitor M’s ECL reagent
were prepared according to the
manufacturer’s instructions. In all three
cases, equal volumes of luminol and
peroxide solutions were combined and
then 4 mL was added to the membranes
and exposed to X-ray film.
Chemiluminescence is routinely involved
in the final stage of detection of proteins
by Western blotting. The most common
and successfully used reaction is
between dihydrophthalazinediones,
such as luminol or isoluminol, an oxidant,
such as hydrogen peroxide and a
peroxidase enzyme, such as HRP. In the
presence of the HRP enzyme and
hydrogen peroxide, luminol is converted
to 3-aminophthalate, via several
intermediates, and is accompanied
by the emission of low intensity light at
428nm (Figure 1).
Low intensity light can be enhanced
>1000-fold with the use of specific
enhancers resulting in simple detection of
the light with film or digital imagers and a
significant increase in sensitivity. This is
more commonly known as Enhanced
Chemiluminescence or ECL.
Experiment 2
Experiment 1
Figure 1: Overview of Western blot chemiluminescence
detection of a protein bound to a membrane. The
protein (antigen) is bound to the membrane and then
a primary antibody that recognises the antigen is
added. A secondary antibody, which specifically binds
to the primary antibody, with horseradish peroxidase
covalently coupled, is then added. Excess antibody
is washed away and then the chemiluminescent
substrates are added. Luminol,
in the presence of hydrogen peroxide (H2O2),
is converted to 3-aminophthalate, via several
intermediates and is accompanied by the emission
of low intensity light at 428nm.
G-Biosciences’ Mouse Normal Liver
GenLysate™ (Cat. No. 82021-808) was
serially diluted and 1, 0.5, 0.25 and
0.125μg total protein was loaded on three
4–20% SDS polyacrylamide gels. The
protein was transferred to PVDF
membranes (Cat. No. 82021-226) in the
presence of Efficient™ Western Transfer
Buffer (Cat. No. 82021-236). The transfer
of the protein to the membranes was
checked with SWIFT™ Membrane Stain
(Cat. No. 89167-886) and following
destaining, the membrane was blocked
Aim
To evaluate picoLUCENT PLUS and compare
its sensitivity and cost to leading
competitor’s ECL reagents.
Method
34
VWR International I VWRbioMarke Issue 1 I April 2015
G-Biosciences’ Cytoplasmic and Nuclear
Cytoplasmic kit (Cat. No. 82022-600) was
used to prepare cytoplasmic and nuclear
fractions from NIH3T3. The protocol was
modified slightly to test different nuclear
protein extraction techniques. 10μg of
each fraction was treated with PAGEPerfect™ (Cat. No. 82021-272) to
concentrate and clean up the fractions.
10μg was loaded on two 4–20% SDS
polyacrylamide gels and the proteins were
resolved and transferred as in Experiment
1. The membranes were probed with a
rabbit polyclonal against PARP-1 (Santa
Cruz Biotechnology, Inc.) at a
concentration of 1:200. ThePARP-1
protein was detected with the same
secondary antibody and G-Biosciences’
picoLUCENT PLUS and Competitor T
Proteomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Figure 2: Comparison of Enhanced
Chemiluminescence (ECL) reagents. Mouse
Normal Liver GenLysate™ was serially diluted and
1, 0.5, 0.25 and 0.125μg total protein was loaded
and resolved on a 4–20% SDS polyacrylamide
gel. The proteins were transferred to a PVDF
membrane. The housekeeper protein actin was
detected with a rabbit polyclonal antibody. The
primary antibody was detected with a secondary
antibody conjugated to HRP. Working solutions
of G-Biosciences’ picoLUCENT PLUS, Competitor
T’s Substrate and Competitor M’s ECL reagent
were prepared according to the manufacturer’s
instructions and used to detect actin. Competitor
M is a highly sensitive reagent with reported
similar sensitivity to G-Biosciences’ picoLUCENT
PLUS.
picoLUCENT™ PLUS
Competitor T
Figure 4: Price comparison of 1L of working
solution. The current USA list price of the three
chemiluminescence reagents tested was compared as
of January 2014. G-Biosciences picoLUCENT was the
more affordable reagent.
Nuclear Extraction ll
Nuclear Extraction l
Cytosol
Nuclear Extraction ll
Nuclear Extraction l
Cytosol
Competitor M
PARP-1
G-BioSciences Competitor T
Actin
Figure 3: Comparison of G-Biosciences and Competitor T picogram chemiluminescence reagents. NIH3T3 cells were
fractionated into nuclear and cytosolic proteins with G-Biosciences’ Cytoplasmic & Nuclear Protein kit (Cat. No.
82022-600). The proteins were resolved on a 4–20% SDS polyacrylamide gel and transferred to a PVDF membrane.
The membranes were probed with a PARP-1 antibody, appropriate secondary antibody and then working solutions of
G-Biosciences’ picoLUCENT PLUS or Competitor T Substrate.
The blots were exposed to film for 1 minute. The blots were then stripped with G-Biosciences’ Western ReProbe™
(Cat. No. 82022-512) and reprobed for actin.
Substrate as in Experiment 1. After
detection of PARP-1 the blots were
stripped with Western ReProbe (Cat. No.
82022-512) and reprobed with actin as in
Experiment 1.
Results & Discussion
Figure 2 clearly shows that the enhanced
formulation of picoLUCENT PLUS is
comparable to Competitor M’s ECL
reagent, whereas Competitor T failed to
adequately detect any actin protein.
Interestingly, the primary antibody used
was a 5-fold higher dilution (1:5000) of the
supplier’s recommended highest dilution
(1:1000) suggesting an improvement in the
sensitivity of the reaction.
Figure 3 also demonstrates a large
difference between the detection levels of
G-Biosciences’ picoLUCENT PLUS and
Competitor T’s substrate. Substrate T is
only able to detect only low level of full
length PARP-1 and no actin.
Figures 2 and 3 demonstrates the
sensitivity of picoLUCENT PLUS and Figure
4 shows how picoLUCENT PLUS is also
more affordable than the leading
competitors products.
References
1. Li, Q. (2009) Reproduction 137:297
2. Maruscak, A., et al (2008) Am J Physiol. Lung Cell Mol.
Physiol., 294: L974
3. Nayak, B. P. et al (2003) J. Virol. 77:10850
Cat. No.
Description
Size
picoLUCENTPLUS HRP
100 mL kit
95043-378
picoLUCENT PLUS-HRP
Chemiluminescent
Reagents Only
100 mL kit
89167-700
20 mL kit
95043-380
200 mL kit
10063-116
500 mL kit
10063-118
1 L kit
10063-124
Nuclear and Cytoplasmic
Extraction Kit
100 preps
82022-600
PAGE-Perfect
50 preps
82021-272
Efficient Western Transfer
Buffer
1L
82021-236
SWIFT Membrane Stain
250 mL
89167-886
NAP-BLOCKER [2X] blocking
agent
1L
82022-626
Western ReProbe [5X]
100 mL
82022-512
April 2015 I VWRbioMarke Issue 1 I VWR International
35
Azure Biosystems Presents the Only Imaging
System for NIR, RGB, and Chemiluminescent
Western Blot Applications
Azure Biosystems is dedicated to designing instruments that can deliver
industry-leading performance across a broad range of laboratory
applications, without overcomplicating the user experience.
We have designed a suite of upgradeable
Western blot imaging systems that give
researchers industry-leading performance
in a flexible system that allows them to use
the best application for their research. Our
instrument product line culminates in the
c600 system (Figure 1), and is the only
imaging line on the market that combines
the following features:
• Infrared laser excitation for quantitative
Western blot imaging in the near IR
• Picogram detection of proteins with
chemiluminescent Westerns
• Versatile dye selection with
Cy®5/Cy3/Cy2 excitation, and more
• Base unit fully upgrageable to the
c600 system
Customers no longer have to choose one
chemistry for their Western blots, they can
12
buy one system and customize it to
their needs.
Imaging in the Near IR
The popularity of near-infrared fluorescent
(NIR) Western blot detection is due in part
to the signal stability and low background
offered by infrared fluorescent dyes, but
more importantly to the additional
questions you can ask with multiplex
fluorescent detection. Azure Biosystem’s
cSeries laser technology offers two IR
detection channels enabling a user to
image more than one protein in an assay.
Imaging with NIR dyes allows you to study
multiple proteins in a blot, even if those
proteins overlap in molecular weight.
Because the secondary antibodies are
imaged in two different channels, the
resulting image can be spectrally
A
A
Figure 1. Azure Biosystems c600 imager. The only
system in the market able to image IR Western blots,
RGB Western blots and chemiluminescent blots.
IR, RGB, and Chemiluminescent Western all in one,
upgradable, cutting edge system.
B
5000 2500 1250 625 313 156
78
39
20
10
5
2.5 pg
Azure, 40 second exposure
B
12
C
Figure 2. Simultaneous detection of EGFR and
phospho-EGFR. Control cells (lane 1) and cells
treated with EGF (lane 2) were imaged. EGFR
was detected in the green channel (panel B), and
phospho-EGFR was detected in the red channel
(panel C). Panel A shows the green and red
channels superimposed.
36
Competitor’s laser scanner system,
5 minute exposure
12
Figure 3. (a) Two color Western blot imaged with IR 700 and IR 800. (b) Azure performs equal to a
competitor’s laser scanner system, 7.5-times faster. A serial dilution of IR 700 antibody shows that the limit of
detection is the same.
VWR International I VWRbioMarke Issue 1 I April 2015
Proteomics
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separated for analysis (Figure 2).
Additionally, you can use your second
channel to easily probe for a loading
control (Figure 3a). Our laser technology
enables sensitivity that meets the
performance of other laser systems
(Figure 3b).
Sensitive Detection of
Chemiluminescent Proteins
Chemiluminescence is still the most
sensitive detection method for some
assays. The Azure cSeries provides accurate
and fast chemiluminescent detection, and
the sensitivity, dynamic range, and linearity
needed for quantatitative blot analysis. No
matter what HRP substrate you are using,
the Azure cSeries systems are compatible
with your current protocol. Instead of using
film and a developer, simply place your blot
in the cSeries to get great results.
Using high resolution cameras, and F 0.95
fast lens techonology, you can capture
images with the same sensitivity as film
(Figure 4a).
A
B
The biggest advantage to switching to
digital imaging is the ability to get more
quantitative data from your Western blots.
Film saturates quickly, making it difficult to
quantify high-abundance proteins. The
Azure cSeries has a broad dynamic range
allowing quantitation over several orders
of magnitude of protein concentration
(Figure 4b).
Versatile Dye Selection
The Azure cSeries is not limited to just NIR
and chemiluminescent Westerns. The
Azure cSeries instruments can also be
equipped with powerful LEDS for Cy5/Cy3/
Cy2 imaging or similar. The system also
contains UV, white, and blue lights for
imaging Ethidium bromide, Coomassie,
and Safe dyes. The cSeries is also able to
image a wide range of dyes that have
excitation maxima from 302nm to 785nm.
This enables WesternDots™, Stain-Free™
gels and different types of in-gel
fluorescence (Figure 5).
Azure cSeries
The Azure cSeries imagers are easy-to-use
and reliable instruments for Western blot
imaging, and enable labs to use one
system for fluorescent and
chemiluminescent Westerns. The simple
user interface allows fast imaging of all
sample types. Full upgradability means
customers can have confidence that their
system will grow and adapt with
their needs.
Description
Cat No.
c200: The Gel Imaging Workstation
10147-222
c300: The Darkroom Replacer cSeries
Imaging System
10147-220
c400: T he Visible Fluorescent Western
Imaging System
10147-218
c500: The Infrared Imaging System
10147-216
c:600 T he Ultimate Western Blot Imaging
System
10147-214
Azure cSeries
35000
Pixel Intensity
30000
R2 = 0.99613
25000
20000
WesternDots®
Stain-Free™
White Light Imaging
Coomassie Blue,
Silver Stain
UV Imaging
Ethidium Bromide
15000
10000
5000
Blue Light Imaging
SYBR® Safe
SYBR® Gold,
SYBR® Green
0
0 5 1015 20 25 3035
Antibody (picograms)
0.5 1
2
4
8
16
32
64 pg
Azure c600
Figure 4. (a) Two slot blots of serially diluted HRP-coupled secondary antibodies were prepared
on nitrocellulose. Both blots were treated with a substrate. Left: Imaged on the Azure cSeries
for 2 minutes. Right: Imaged on film for 2 minutes. (b) Azure cSeries gives a linear response to a
serial dilution of an HRP-coupled antibody.
Figure 5. Application flexibility. The Azure c600 has eight
different light sources and a seven position filter wheel,
resulting in compatibility for the most critical dyes for
protein work.
April 2015 I VWRbioMarke Issue 1 I VWR International
37
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
The New Thermo Scientific™
Pierce™ Power Stainer
A Fast, Reliable Device for Coomassie Protein Staining
The Pierce Power Stainer is an easy to use device for rapid electrophoretic
Coomassie staining and destaining of protein gels in 6–11 minutes with
equivalent, or better staining performance relative to traditional solutionbased Coomassie staining.
Protein staining represents an important
and ubiquitous technique in life science
research where traditional Coomassie
staining requires relatively long incubations
of the polyacrylamide gel in stain and
destain solutions. The new electrophoretic
method uses the Pierce Power Station, an
integrated high current power supply unit with
PIERCE
POWER STATION CASSETTE
Cathode (-)
Staining pad
Gel
Destaining pad
Anode (+)
Figure 1. The Pierce Power Stainer is
designed for rapid Coomassie staining
of proteins in polyacrylamide gels and
removal of unbound stain from the
gel (i.e., destaining) in a single step.
Traditional Coomassie staining techniques
require a 1-hour to overnight staining
step and a separate destaining step(s)
for desired results. With the use of the
Thermo Scientific Pierce Power Staining
Kits (PI22839, PI22840), the Pierce Power
Stainer (PI22833) provides efficient
protein staining and gel destaining in
6 – 11 minutes producing results
equivalent to, or better than, traditional
Coomassie staining techniques. The
Thermo Scientific Pierce Power Station
(PI22838) component of the Power
Stainer has an easy-to-use color
LCD touchscreen interface and preprogrammed gel staining protocols. The
easy-touch programming feature allows
custom staining settings to be quickly
created, saved, and run.
38
VWR International I VWRbioMarke Issue 1 I April 2015
staining reagents and consumables.
Optimization of the reagents with
electrophoretic staining and destaining
conditions (current, voltage, and time) results in
excellent staining performance with commonly
used pre-cast and homemade SDS-PAGE gels,
including Bolt®, NuPAGE®
and Novex® gels.
Proteomics
MW
5ng
15ng
45ng
135ng
405ng
MW
5ng
15ng
45ng
135ng
C.
405ng
5ng
15ng
45ng
135ng
405ng
B.
MW
A.
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
200
120 150
100 85
70 60
50
40
30
25
20
MW
5ng
15ng
45ng
135ng
405ng
MW
5ng
45ng
15ng
C.
135ng
405ng
5ng
15ng
45ng
135ng
405ng
B.
MW
A.
200
120 150
100 85
70 60
Pierce Power Stainer
1. Water wash (1 x 5 min)
2. Staining (6 min)
Total
protocol
Pierce Power
time: Stainer
1. Water wash (1 x 5 min)
2. Staining (6 min)
Total
protocol
time:
50
Imperial
40
Basic Coomassie Stain
1. Water wash (3 x 5 min)
2. Stain (60 min)
3. Destain (overnight)
4. Water wash (4 x 15 min)
Basic Coomassie Stain
~11 minutes
10
Protein Stain
30
1. Water
wash (3 x 5 min)
25
20
2. Stain
(60 min)
3. Water
wash (3 x 20 min)
15
4. Water
wash (overnight)
10
~18Imperial
hours Protein Stain
1. Water wash (3 x 5 min)
2. Stain (60 min)
3. Destain (overnight)
4. Water wash (4 x 15 min)
1. Water wash (3 x 5 min)
2. Stain (60 min)
3. Water wash (3 x 20 min)
4. Water wash (overnight)
~18 hours
~18 hours
~11 minutes
15
~18 hours
Figure 2. Thermo Scientific Pierce Power Stainer saves time and maintains sensitivity. Three
4-20% Tris-Glycine mini gels were loaded and run with samples of purified bovine serum
albumin and staining protocols compared for time. A. Gel was stained using the Pierce
Power Stainer (PI22833) and the Pierce Mini Gel Power Staining Kit (PI22840). B. Gel was
stained using a homebrew Coomassie R250 staining protocol. (Stain: 45% methanol, 10%
acetic acid, 0.25% R-250 Coomassie; Destain: 30% ethanol, 5% acetic acid) C. Another
gel was stained with Imperial Protein Stain using manufacturer’s instructions. Cell lysate,
purified proteins and unstained molecular weight markers were loaded on Novex 4-12%
Tris-Glycine gels and electrophoresed according to the gel suppliers’ recommendations.
After electrophoresis, one gel was stained using Thermo Scientific Imperial Protein Stain
(PI24615) by first washing the gel three times 10 minutes with ultra pure water, staining
Cathode (-)
forStaining
60 minutes
in the stain and destaining overnight in water. The second gel was stained
pad
Gel
using
Pierce
Power Stainer and Mini-Gel Power Staining Kit for 7 minutes. The third gel was
Destaining
pad
Anode (+) using the staining device and consumables from supplier A for 7 minutes according
stained
to the manufacturer’s instructions.
Cathode (-)
Staining pad
Gel
Destaining pad
Anode (+)
Mini-Protean 4-20% TGX Gel
5 min
Criterion 4-20% Tris-HCI Gel
10 min
Novex 4-20% Tris-Glycine Midi Gel
10 min
Novex Bolt 4-12% Bis-Tris Gel
5 min
Novex NuPAGE 4-12% Bis-Tris Midi Gel
10 min
Home-Made 10% Tris-Glycine Mini Gel
6 min 30 sec
Figure 3. The Pierce Power Stainer is compatible with multiple gel chemistries. The
performance of the Pierce Power Stainer (PI22833) was tested with different brands precast gels including Novex, NuPAGE, Bolt, Criterion, and Mini-Protean as well as homemade
tris-glycine gels. Cell lysate, purified proteins, and unstained molecular weight markers
were loaded on different mini-sized and midi-sized protein gels and electrophoresed
according to the gel suppliers’ recommendations. After electrophoresis, the gels were
washed in water one time for 5 minutes (mini-sized gels) or two times for 5 minutes
(midi-sized gels) and then stained using Pierce Power Stainer and Mini-Gel Power Staining
Kit (PI22840) or Midi-Gel Power Staining Kit (PI22839) for 5–10 minutes. The Pierce
Power Stainer is compatible with all the gel chemistries tested and provided excellent
protein staining results with sharply stained protein bands with little or no background.
Additionally, 1.5mm thick gels (NuPAGE and Tris-Glycine) were found to work with the
Pierce Power Stainer (data not shown).
Description
Cat. No.
Pierce Power Stainer
PI22833
Pierce Power Blotter
PI22834
Pierce Power System
PI22830
Pierce G2 Fast Blotter – Power Stainer Upgrade
PI62287
Features and benefits:
•
Fast – Coomassie staining and destaining of proteins in
polyacrylamide gels in just 6–11 minutes (less than 20 minutes total
protocol time)
•
Easy to Use – Intuitive, color LCD touchscreen interface and preprogrammed staining methods. Software includes “Learn as you
run” tutorials
•
High quality results – The integrated power supply and the reagents
are optimized to deliver efficient staining equivalent to, or better
than, traditional Coomassie staining techniques
•
Cost Effective – Cost for consumables is comparable to other
commercial solution-based staining (~$3.80/mini gel)
•
Flexible – Works with pre-cast and homemade SDS-PAGE gels
•
Convenient – Simultaneous staining and destaining of 1 to 2 minisized gels or 1 midi-sized gel
•
•
Optimized Accessories – Gel pads included in the Power Staining
Kits (PI22839, PI22840) enable uniform staining and minimize
blotchy background staining
Upgrade-ready – Adding the Pierce Power Blot Cassette
(PI22835) activates the pre-loaded software making this unit a
fully functional Pierce Power System (PI22830) with blotting and
gel staining capabilities.
April 2015 I VWRbioMarke Issue 1 I VWR International
39
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
Multiplex Fluorescent Western Blot Detection
Using the BioSpectrum Imaging System
Abhishek Trikha, BVSc & A.H , M.S. and Samet Serdar Yildirim, MSc, PSM
UVP LLC, Upland, CA
Introduction
Western blotting is a commonly used
analytical technique for the identification
and quantification of specific proteins in a
biological sample. Traditionally, a target
protein is interrogated by antigen-specific
primary antibodies which are then probed
by secondary antibodies conjugated to
either Horseradish Peroxidase (HRP) or
Alkaline Phosphatase (ALP) and followed by
colorimetric or chemiluminescent detection.
Fluorescent Western blotting employs
secondary antibodies labeled with a
fluorophore to perform non-enzymatic
detection of protein expression. On an
immunoblot incubated with two different
primary antibodies from different species
and then probed with CyDye™-tagged
secondary antibodies for detection (Figure
1), the two-color multiplexing abilities of
the BioSpectrum Imaging System with the
BioLite Xe MultiSpectral Light Source allow
for detection of multiple proteins.
Additionally, fluorescent blotting offers
excellent signal stability over time, as well as
accurate quantitative analysis with broader
dynamic range and high linearity, thus
reducing or eliminating the need to strip
and re-probe.
Figure 1. Schematic of Fluorescent Western Blotting
Materials and Methods
Sample Preparation and
Western Blotting
Two-fold dilutions of normal rabbit and
mouse sera (Jackson Immuno Research
Laboratories) were separated by SDS PAGE
on 4-12% acrylamide gels (Invitrogen). The
separated proteins were then transferred
to nitrocellulose membranes.1
Blots were simultaneously probed with
goat-α-rabbit IgG–Cy®3 and goat-α-mouse
IgG–Cy5 (GE Healthcare Life Sciences)
secondary antibodies at 1:1250
concentration in Western Breeze blocking
buffer (Invitrogen) for one hour incubation
at room temperature. Blots were washed
in secondary antibody
incubations with 4 x 5 minutes in trisbuffered saline (TBS) containing 0.1%
Tween® 20.
Imaging
The BioSpectrum Imaging System with the
BioLite Xe MultiSpectral Light Source (UVP,
LLC) was used for fluorescent imaging
(Figure 3). Images were processed with
VisionWorks®LS Image Acquisition and
Analysis software (UVP, LLC) to remove
background intensity and composite the
pseudocolored images.
The processed blot was positioned on the
sample platen. Utilizing software presets,
the excitation and emission wavelengths
were selected, the lens aperture was set at
f/1.2, and the image was focused. Exposure
time was adjusted for maximal signal
without saturation and ranged from 1 to 5
seconds, depending on the sample and
filter set (Table 1). Once acquired, the
original unaltered image was archived and
a copy was used for image analysis. The
image was adjusted to globally remove
background intensity and contrast, and was
40
VWR International I VWRbioMarke Issue 1 I April 2015
Proteomics
For more information on these products, visit vwr.com,
call 800.932.5000, or contact your VWR representative.
ProteOmics
A
B
C
Figure 2. Multiplex Fluorescent Western Blot Detection with CyDyes using BioSpectrum Imaging System
a) Rabbit IgG probed with Cy3–tagged goat anti-rabbit IgG b) Mouse IgG probed with Cy5-tagged goat anti-mouse IgG c) Multiplex fluorescent detection of two
fold serial dilution of mouse and rabbit serum proteins probed with Cy3–tagged goat anti-rabbit IgG and Cy5-tagged goat anti-mouse IgG, respectively, on same
immunoblot. CyDyes were imaged using a one second exposure with the BioSpectrum Imaging System.
Figure 3. BioSpectrum Connected to the BioLite
Together, the BioSpectrum and BioLite are a
powerful combination that are able to specifically
excite and illuminate at wavelengths from 365 to
765nm and read emissions from 400 to 850nm.
Use of up to eight excitation and five emission
wavelengths are possible in a single experiment.
The BioSpectrum 810 and BioSpectrum 610 are
recommended for imaging multiplex fluorescent
Western blots.
Dye
Excitation
Emission
Cy3
525BP45
605BP50
Cy5
630BP30
730BP40
Table 1. Filters used for NIR blotting with 680 and 770
to 800nm fluorescent tags.
Imaging Time
Compared to laser scanning imaging
systems where imaging times range
from 3 to 5 minutes, the BioSpectrum
Imaging System provides a significant
advantage with imaging times ranging
from 1 to 5 seconds for fluorescent
Western blotting applications.
Conclusion
pseudocolored green and red using
VisionWorksLS Software to indicate Cy3 and
Cy5, respectively.
Results and Discussion
Figure 2 illustrates the multiplex imaging
capabilities of the BioSpectrum Imaging
System, specifically separating out the
signal of both Cy3 and Cy5 color channels.
Fluorescent Western blotting applications
offer many advantages over chemiluminescent or chromogenic visualization.
Most significantly, fluorescent labels
permit multiplexing so that several
proteins in a sample can be detected and
analyzed at the same time and on a single
protein blot. Fluorescent labels in particular
offer very low background and a high
signal-to-noise ratio for quantitative
imaging. The combination of the
BioSpectrum Imaging System and the
BioLite MultiSpectral Light Source provides
not only a full range of wavelengths for
excitation light but also rapid high
resolution image capture through the use
of deeply cooled MegaCam 810 and
OptiChemi™ 610 cameras and low
light lenses.
Fluorescent Western blot imaging with the
BioSpectrum Imaging System and BioLite
MultiSpectral Light Source is a fast and
efficient process for enabling researchers
to achieve simultaneous detection of
multiple proteins on the same immunoblot
and generate high resolution, publicationready images.
References
1. Gallagher, S.R. and Wiley, E.A. Current Protocols: Essential
Laboratory Techniques. Wiley, 2012
Description
Biospectrum Imaging System
Cat. No.
10551-278
April 2015 I VWRbioMarke Issue 1 I VWR International
41
Inside the Biotix
Robotic Laboratory
With the continuing trend
of labs incorporating liquid
handling automation
into their workflows,
performance and quality
are top considerations
when selecting the right
pipette tips to use with
these systems. For high
throughput automation,
robotic tips are expected
to meet stringent quality
manufacturing standards.
QC processes must
ensure batch-to-batch
conformity as well as meet
exacting requirements
for straightness,
sealing, purity, accuracy
and precision.
42
In order to manufacture robotic tips to the highest
engineering standards for automation systems,
Biotix has built an in-house Robotic Laboratory as
a key element of its QC process for validation and
release testing. Unlike other manufacturers that
only perform dimensional inspection to
engineering drawings, Biotix requires both
dimensional and functional testing. A
combination of specialized fixtures and functional
application tests are executed using the specific
automated liquid handling system that the tips
have been designed for. Experienced technicians
run the products through a world class quality
system measuring coefficient of variance (CV),
total indicated run out (TIR), insertion force and
many other performance tests on the robots. If a
product fails any test, it is rejected, ensuring that
only the highest quality tips reach the end
user’s lab.
As a result, Biotix ultra-straight tips target the
centre of microplate wells precisely, delivering
exceptional accuracy and precision without risk
of jamming, offering the highest quality robotic
VWR International I VWRbioMarke Issue 1 I April 2015
tips the industry has ever seen with specifications
to ensure complete compatibility. Use of the
standard definitions bundled in the robotic
software program is appropriate when using
Biotix robotic tips. These tips are also BioReady™
certified to be free of RNase, DNase, and
endotoxins (pyrogens).
Biotix robotic tips are compatible with these
popular brands of automated liquid
handling systems:
Agilent® Beckman®
®
Tecan Caliper®
®
BioTek PerkinElmer/Packard®
®
Molecular Devices
For more information on Biotix robotic tips,
contact your local VWR Representative or
visit vwr.com.
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VWR has proven expertise in providing you with product and service solutions
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From biochemicals and kits to serum for research and scale up production,
the VWR Life Science brand supports innovation, so you can focus on what’s
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scientific innovation — from research to production.
April 2015 I VWRbioMarke Issue 1 I VWR International
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