Western in A Day
Ning Tingting, Ph.D
Field Application Specialist, BIO-RAD
Tuesday, March 22, 2016
印记法
Northern Blot
RNA
Protein
DNA
Western Blot
Far Western
Blot
Dot Blot
Southern Blot
Transfer Methods
The principle of Electrophoretic transfer
Proteins migrate to the membrane following a current (I) that is
generated by applying a voltage (V) across the electrodes,
following Ohm’s law:
V=IxR
Transfer Methods
The principle of Electrophoretic transfer
1. the applied voltage and the distance between the
electrodes play a major role in governing the rate of elution
of the proteins from the gel.
2. the size, shape, and charge of the protein and the pH,
viscosity, and ionic strength of the transfer buffer and gel
%T influence the elution of particular proteins from gels
There are practical limits on field strength, however, due to the
production of heat during transfer.
Joule heating is proportional to P = I x V = I2 x R
Transfer Methods
The principle of Electrophoretic transfer
Joule heating is proportional to P = I x V = I2 x R
Joule heating 
• Buffer resistance 
• Buffer buffering capacity 
• Inconsistent field strength and transfer
• Gel to deteriorate and stick to the membrane
The major limitation of any electrophoretic transfer method
is the ability of the chamber to dissipate heat
Transfer Methods
Types of Electrophoretic transfer
Tank Blotting
Semi-Dry Blotting
Flexibility
Flexible voltage settings, blotting times, and
cooling requirements; flexible electrode
positions (Trans-Blot and Trans-Blot Plus cells)
(Trans-Blot and Trans-Blot Plus cells)
without cooling
Quantitative vs
qualitative results
Quantitative transfer of low molecular weight
proteins possible under conditions that allow
efficient binding to the membrane
Some low molecular weight molecules will
be transferred through the membrane
without binding quantitatively
Molecular weight
range
Broad molecular weight range
Variable transfer efficiencies for proteins
>120 kD (may be improved with
discontinuous buffer system); low
molecular weight proteins may be
transferred through membrane
Transfer time
Extended transfer (up to 24 hr) possible without
buffer depletion; rapid transfers (15–60 min)
obtained under high-intensity conditions
Rapid transfers; extended transfers not
possible due to buffer depletion
Temperature
control
Specific temperature regulation with cooling
coil and refrigerated water recirculator; permits
transfers at low temperatures (4–10°C), for
example, native enzyme transfers
Temperature regulation by external cooling
is not possible
Buffer capacity
Up to 10–12 L (Trans-Blot Plus cell) or as little
as 450 ml (Mini Trans-Blot cell); length of
blotting time not restricted by limited buffer
capacity
Minimal, ~250 ml per experiment; reduced
cost of reagents and experiment time
Membrane, Buffer & Power conditions
Membrane selection
Membrane
Nitrocellulose
Supported Nitrocellulose
Pore size
0.45 µm
0.2 µm
0.45 µm
0.2 µm
Binding capacity Comments
(µg/cm2)
80-100
good general purpose blotting membrane
80-100
Pure nitrocellulose cast on an inert synthetic
support; increased strength for easier handling
and for reprobing
Sequi Blot PVDF
0.2 µm
170-200
High mechanical strength and chemical stability;
high binding capacity, also with SDS
used for protein sequencing of even low abundance
sample
Immun Blot PVDF
0.2 µm
150-160
High mechanical strength and chemical stability;
high binding capacity, also with SDS
used for protein immunodetection
! PVDF membrane must be wetted in 100% methanol prior to use but may be used
with a transfer buffer that contains no methanol
Membrane, Buffer & Power conditions
Transfer buffer selection
General recommandation:
SDS in buffer:
protein mobility
binding efficiency to membrane
– SDS confers a negative charge to positive and neutral proteins
– increases transfer efficiency of large proteins
– recommended when using SDS: PVDF or positively charged nylon
membranes
– will increase the relative current, power and heating
– may affect the antigenicity of some proteins
– à discontinuous system with only 0.01 % SDS
Membrane, Buffer & Power conditions
Transfer buffer selection
General recommandation:
Methanol in buffer:
 protein transfer from gel
binding to nitrocellulose
– not necessary with PVDF or nylon membranes
– gel shrinks, the pores of the gel are reduced
– removes SDS from SDS-protein complexes
– precipitation, denaturing, loss of biologic activity
Membrane, Buffer & Power conditions
Transfer buffer selection
Standard Towbin buffer contains 25 mM Tris, pH 8.3, 192 mM glycine, 20% (v/v)
methanol and, occasionally, 0.025–0.1% (w/v) SDS.
A buffer similar in composition to the standard Towbin buffer is the Bjerrum and
Schafer-Nielsen buffer (48 mM Tris, pH 9.2, 39 mM glycine, 20% methanol),
which was developed for use in semi-dry applications
CAPS buffers (10 mM CAPS, pH 11, 10% methanol) may be preferable for
transfers of high molecular weight proteins (for example, >150 kD) and in cases
where the glycine component of Towbin buffer may interfere with downstream
protein sequencing applications
Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9, 20%
methanol) may produce higher efficiency transfers and improve the ability of
antibodies to recognize and bind to proteins
Membrane, Buffer & Power conditions
Transfer buffer selection
Discontinuous Tris-CAPS Buffer System (Semi-Dry Transfers)
60 mM Tris, 40 mM CAPS, pH 9.6, plus 0.1% SDS
60 mM Tris, 40 mM CAPS, pH 9.6, plus 15% methanol
Membrane, Buffer & Power conditions
Power conditions for Electrophoretic Transfers
Selecting power supply settings
During tansfer, the resistance decreases as a result of Joule heating,
except for semi dry blotting where R increases due to ion depletion.
Transfer under constant voltage
U cst = R x I 
-need a cooling system
- field strength will remain constant, providing the most efficient transfer possible
Transfer under constant current
U = R x I cst
-heating minimized
- proteins will be transferred more slowly due to decreased field strength
Transfer under constant power
P cst = R  x I2  = U2  / R 
-current increases but less than for Vcst (√), decreased field strength
- alternative to constant current for regulating heat production during transfer
Performing the transfer
Tips
•Remove all air bubbles at each step with a roller
•To avoid ghost prints and other artifacts, do not move the membrane and/or gel
after it is positioned
•Place the transfer tank onto a magnetic stirplate
•The tanks are effective thermal insulators and limit the efficient dissipation of heat.
Therefore, placing blotting cells in a coldroom is not an adequate means of
controlling transfer buffer temperature. Use the internal cooling devices
•For SD, use one extra thick filter paper instead of two or three thin or thick filter
paper to avoid air bubbles between them
•For SD, the membrane and filter paper should be cut to the same size as the gel
Detection
Detection
Detection Methods
Detection
Immunological Detection Procedure
Detection
Immunological detection methods
A’. Colorimetric amplified
AP detection
What is important to a western blot
experiment?
• Can I see my target proteins?
– if not, what’s wrong?
• Transfer
• Sample
• Detection sensitivity
– if yes, how reliable?
• Accurate?
• Reproducible?
• Can I get away from the tedious work?
“Bio-Rad V3 WB workflow” to address
these issues
• Can I see my target proteins?
– if not, what’s wrong?
• Transfer (Verify better monitoring of transfer efficiency)
• Sample (Visualize sample quality control)
• Detection sensitivity (more sensitive detection reagents)
– if yes, how reliable?
• Accurate? (Validate – Quantification and loading control)
• Reproducible? (transparency of the whole process for better
monitoring, such as loading error, uneven transfer etc.)
• Can I get away from the tedious work?
Fast procedure
Multiplexing
Content
•
What is “Bio-Rad V3 Western Blot Workflow”
•
What is important to a western blot experiment?
•
Visualize gel run
•
Verify Transfer efficiency
•
Validate quantitative analysis
•
Multiplexing of the detection
•
Comparison between Bio-Rad “Bio-Rad V3 Western Blot Workflow” and
the traditional western blot approach
What is it?
Bio-Rad V3 Western Blot workflow is a Bio-Rad
complete western blot solution incorporated with
new technologies and products, such as TGX stain
free gels, Trans-blot Turbo and ChemiDoc MP imager
system, to provide a best practice in western blot
experiment to ensure higher confidence in the
experimental findings over the traditional western
blot workflow.
The traditional western blot workflow
Traditional WB Approach
Gel prep
>1h
By ChemiDoc MP
(optional view under UV)
~1h
Electrophoresis
1-3 h
blotting
Immunodetection
~5h
Imaging & data
analysis
>30min
Often need reprobing ~5h
By eye
Loading control
reprobing
Visualize
眼见为实
Bio-Rad Stain Free Technology
ChemiDoc MP system
Stain Free Pre-cast Gels
Stain Free Technology
1. Stain-free compound is premixed with the gel, therefore no staining
step required
2. Gel image can be generated 5 minutes after electrophoresis is
finished
3. UV induced fluorescent gel or blot image
4. Low background produced and staining is uniform across the gel
and blot
5. Non-reversible staining on both the gel and blot (can not wash
away)
6. The same gel is used for both total protein loading control and
transfer for western blotting
Sample quality control:
Stain Free Technology to Visualize the gels
within 5 min after gel run
Fraction #
1
2
3
4
C C A A C C A A C C A A C C A A
T.P.
75 kDa
50 kDa
25 kDa
A total protein gel image allows a double check of the protein
profiles in each sample to avoid artifacts caused by inaccurate
protein assay and/or protein degradations.
The sample quality control tells you when to terminate your
experiment… Don’t waste time and money on bad samples
Verify
确认转印
Fast Blotting of TGX Stain Free gels make
it easy to verify transfer efficiency
3 Min
Trans-Blot Turbo with TGX gels
15 Min
Tank Blotting with TGX gels
How do I know transfer was successful
and how to optimize it?
1. Traditional approach
• only indicative, prestained protein standards
• no image recording, not quantitatively
2. Bio-Rad V3 WB approach
• quantitative Stain Free Gel image
• true visualization of the protein samples
3. To optimize transfer conditions
1. easy and fast
2. If some proteins remained in the gel after transfer, set up another
blotting with the same gel to give another 5 min of transfer
3. Add this extra time to the original setting for next blotting experiment
Now you can see proteins on
the gels before transfer
Stain free image of protein samples on the Gel
Double check your loading consistency
Double check the sample quality
Now you can see proteins on
the post-transfer gels
Stain free image of a Post-Blot Gel
No more guess about the transfer efficiency.
Now you have the control – you can calculate how many percent of
the total protein are still left in the post-transfer gel…
Now you can see proteins on the blot
Stain Free image of proteins on a blot
Worry about protein loss during striping for reprobing?
No more worries, now you have the control – simply put the blot in
the ChemiDoc MP to take a quick look…
Validation
定量校验
Linear dynamic ranges
Loading control
Western Blot can be more quantitative
Introduction to Western Blot Validation

Validation
of western blot results: Why and How?
 How?
Typically, a specific housekeeping protein is used as
loading control (GAPDH, Actin, Tubulin…)
 Experimental
errors are identified by quantitative differences in HKP
 Target protein data is normalized to the HKP signal
 Most customers do chemiluminescence detection → Strip and reprobe or cut
the membrane
 Sounds easy, right? What‘s the problem with both experiments below?
Experiment B (same publication!)
Experiment A
What is loading control?
What are the commonly used loading control
proteins?
Samples of loading control in publication -PNAS
Fig. 7. Western blots showing the extents of
calmodulin over-expression in DG75 cells (A) and
splenic B-cells (B). The levels of calmodulin in
nuclear and cytoplasmic extracts of calmodulin
over-expression experiments with and without antiIgM treatment in DG75 cells and in splenic B-cells
(Figs. 2A and 5G, respectively) were determined
by Western blot analysis. The Western blots were
performed using the WesternBreeze
immunodetection system (Invitrogen) according to
the manufacturer's instructions, using calmodulin
antibody (FL-149; Santa Cruz Biotechnology).
Tubulin antibody (clone B-5-1-2; Sigma-Aldrich)
was used as loading control for cytoplasmic
extracts and histone H1 antibody (FL-219; Santa
Cruz Biotechnology) as loading control for
nuclear extracts.
What’s wrong?
What’s wrong?
What’s wrong?
Western Blotting Help!!!! - Problem with an internal control (Jul/28/2004 )
Hello ALL!
I’m hoping someone can explain what is going on with some of my data!!!!
We are currently checking cell viability post cell explant from saphenous vein tissue. Our
viability data indicates that we are isolating less cells from the tissue as time increases and
that these cells in culture are less viable. So to look at specific cell markers we thought it
locgical to run our samples on a SDS-PAGE and use Beta Actin as a loading control. Time after
time we are loading equal amounts of protein per lane but our cell specific marker is
decreasing (which is logical...that is the amount of cells expressing H-caldesmon.....) but our
B-actin which should be ubiquitous to all cells is also decreasing at a similar ratio. This doesnt
make sense to me as we were hoping to see loss of protein cell-specific protein, only!! We
have used BCA to measure our protein concentations in our cell lysates, and have used kits
from different manufacturers with similar results. We have used different antibodies, both
monoclonal and polclonal. We have also used different H-caldesmon antibodies. Each time
with similar results. Could cell death (which we know by our viability experiments) contribute
to the decrease in beta-actin (our internal control) despite the loading of equal amounts of
protein. By the way, this has also been done with GAPDH and tubulin. I'm at a loss and cant
explain this.
PLEASE HELP?!!!
Doubt
ELECTROPHORESIS Volume 27, Issue 14, pages 2844–2845, No. 14 July 2006
β-Actin is not a reliable loading control in Western blot analysis
Angela Dittmer, Jürgen Dittmer Dr.
Abstract
β-Actin is often used as a loading control in Western blot analysis. We
analyzed the ability of β-actin-specific antibodies to recognize
differences in protein loading. We found that, at higher total protein
loads as required for the detection of low-abundance proteins, β-actinspecific antibodies failed to distinguish differences in actin protein
levels. Diluting the antibody working solution or changing the incubation
time had little effect on this phenomenon. This shows that β-Actin is not
a reliable loading control in Western blot analysis. In general, it
appeared that, at longer incubation times, antibodies seem to be less
able to pick up differences in the level of its target protein.
Doubt
β-actin varies in some situations
• Varying expression level of β-actin in response
to biomedical stimuli
• Altered expression of β-actin during growth and
differentiation
• Changed expression of β-actin in some diseases
β-actin varies in some situations
Expression difference @PCR level
Reason 2 – overexposure
In the case of the polyclonal anti-b-actin antibody, no change in band
intensity could be observed in the range of 7.5–1.88 mg of total protein.
At 0.94 mg protein the signal gradually decreased and was undectable
at 0.12 mg or lower. The monoclonal anti-b-actin antibody generated a
similar picture, except that similar band intensities were found in a
broader range of protein loads between 0.47 and 7.5 mg.
What are the alternatives for loading control?
What are the alternatives for loading control?
What are the alternatives for loading control?
Option 1- Irreversible
•
•
•
•
Coomassie staining
Silver staining
Amido black staining
Fluoresce staining
Option 2 - reversible
Analytical Biochemistry Volume 401, Issue 2, 15 June 2010, Pages 318-320
Reversible Ponceau staining as a loading control alternative to actin in
Western blots
Isabel Romero-Calvoa, Borja Ocóna, Patricia Martínez-Moyaa, María Dolores Suáreza, Antonio Zarzueloa,
Olga Martínez-Augustina and Fermín Sánchez de Medina , a, a
Departments of Biochemistry and Molecular Biology II and Pharmacology, School of Pharmacy, University
of Granada, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas
(CIBERehd), Campus de Cartuja s/n, 18071 Granada, Spain
Abstract
It is becoming standard practice to measure a housekeeping gene, typically actin, in Western blots, as it is
the rule in RNA blots. We have applied reversible Ponceau staining to check equal loading of gels and
measured actin in parallel under different conditions. Our results show that densitometric analysis is
comparable with both techniques. Therefore, routine quantitation of Ponceau staining before antibody
probing is validated as an alternative to actin blotting.
• Loss of target, Waste of membrane and time
How to measure total protein local on a stain
free image
1. Acquire a stain free gel image
2. Using the Image Lab software “Volume Tools” to define a volume
area to cover the whole lane of a sample
3. Copy and paste the defined the area to cover other sample lanes
4. The total protein amount is measured by the “Adj. Vol.” in the data
analysis table
1 2
3
4
5
6
7
Bio-Rad solution to a more reliable
loading control
TGX Stain Free Precast Gels & ChemiDoc MP Imager System
provide The best way to measure total proteins for loading
controls in a western blot experiment
ChemiDoc MP system
Stain Free Pre-cast Gels
Benefits of the “Bio-Rad V3 WB Workflow”
over traditional approaches
Faster and More Reliable WB workflow
Traditional WB Approach
Gel prep
Precast TGX stain free gel
>1h
~1h
15-20 min
Electrophoresis
Stain Free Imaging
1-3 h
ChemiDoc MP
blotting
Turbo
More reliable
loading control
3-15 min
Sample quality
control
Immunodetection
~5h
~5h
Transfer
optimization
Imaging & data
analysis
ChemiDoc MP <30min
>30min
Protein
visualization on
blot
Multiplex WB
Often need reprobing ~5h
Loading control
reprobing
No need to reprobe
Multiplexing WB to analyze
phosphoproteins
Nature Methods 2, 79 - 81 (2005)
Western blot analysis with quantum dot fluorescence technology:
a sensitive and quantitative method for multiplexed proteomics
Western blot images of p42 MAPK (a, green) and phosphorylated p42 MAPK (b, red) expression in
serum-starved 3T3 cells in response to PDGF stimulation. Cells were harvested at 0, 10, 20, 40
and 60 min after PDGF administration (lanes 1–5, respectively). The anti-pan MAPK primary
antibody (a) was detected with Qdot 605 nm Conjugate, a red-orange dot that was pseudocolored
green and the phospho-MAPK primary antibody (b) was detected with Qdot 705 nm Conjugate and
pseudocolored red. (c) Overlay of images in a and b.
Multiplexing WB to detect 3 different targets
on the same blot using Qdot Conjugates
Excitation: 415nm /100nm filter
Green : Anti-GST , 565nm
Red:
Anti-GST-HA, (rabbit) , 605nm,
Blue:
Anti-GST-cMyc (mouse) 705nm
Multiplexing WB to detect 4 different
targets on the same blot
Hsc70 (green), beta-actin (red) and ERX1 and ERX2 (blue)
Titration of lac operator DNA with lac repressor protein. Increasing amounts of lac
repressor protein were mixed with 40 ng of lac operator DNA, incubated for 20 minutes
and then separated on a 6% nondenaturing PAGE. The gel was stained with SYBR®
Green EMSA stain (green) followed by SYPRO® Ruby EMSA stain (red), components
of the Electrophoretic Mobility-Shift Assay Kit . After each staining, the image was
documented using an FLA-3000 laser-based scanner (Fuji) and the digital images
pseudocolored and overlaid. Yellow bands indicate areas stained with both stains.
This fluorescent western blot shows simultaneous
detection of unphosphorylated and
phosphorylated Akt1 present in serum starved and
PDGF stimulated NIH/3T3 whole cell lysates.
Lane 1, unstimulated NIH/3T3 lysates contain inactive
unphosphorylated Akt1, green band. Lane
2, PDGF stimulated NIH/3T3 lysate contains both
inactive (green band) and activated
phosphorylated Akt1 (red band). Both lanes were
probed with rabbit anti-Akt (pan) and mouse
anti-Akt pS473 specific antibodies followed by
detection with DyLight™ 549 conjugated anti-rabbit
IgG (green) and DyLight™ 649 conjugated anti-mouse
IgG (red) secondary antibodies.
The “Bio-Rad V3 WB workflow”
• Can I see my target proteins?
– if not, what’s wrong?
• Better monitoring of transfer efficiency – stain free gels and
ChemiDoc MP imager
• Sample quality control – stain free gels and ChemiDoc MP imager
• More sensitive detection reagents –ChemiDoc MP imager and
WesternC Chemiluminescence kit
– if yes, how reliable?
• Loading control – stain free gels and ChemiDoc MP imager
• transparency of the whole process for better monitoring – ChemiDoc
MP imager, stain free gels and low fluorescence PVDF membrane
• Can I get away from the tedious work? (fast)
– TGX gels and Trans Blot Turbo
– Fluorescent multiplexing on ChemiDoc MP imager
联系方式：
宁婷婷
Email：Tingting_ning@bio-rad.com
电话：027－83806255－805
移动电话：18627760323