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Western Blotting Troubleshooting Handbook
Western Blotting Troubleshooting Handbook
Introduction .......................................................................................................................... 2
Principle ............................................................................................................................... 2
1. Protein Extraction and Quantitation ............................................................................................. 2
2. Protein Separation: Electrophoresis.............................................................................................. 3
3. Protein Transfer ........................................................................................................................... 5
4. Membrane Blocking ..................................................................................................................... 7
5. Primary Antibody Incubation ........................................................................................................ 8
6. Secondary Antibody Incubation .................................................................................................... 8
7. Signal Detection ........................................................................................................................... 8
8. Controls ..................................................................................................................................... 11
Recommended Protocol ....................................................................................................... 14
1. Sample Preparation ................................................................................................................... 14
2. Electrophoresis .......................................................................................................................... 16
Tips for Convincing Your PI to Purchase Pre-Cast Gels ....................................................... 24
Safety and Good Laboratory Practices .................................................................................. 25
Troubleshooting Guide ........................................................................................................ 26
High Background ........................................................................................................................ 26
Weak/No Signal ......................................................................................................................... 28
Nonspecific Bands ...................................................................................................................... 30
Diffuse Bands ............................................................................................................................. 31
FAQs ................................................................................................................................... 32
Ordering Information ........................................................................................................... 36
Antibody Protein Target Index ............................................................................................ 40
Contact Information ............................................................................................................ 45
1
Introduction
Principle
To extract protein for the western blot analysis, the first step is to prepare for the lysate from samples. Cell
cultures and tissue are the most common samples to start with. When preparing samples for Western blot
it is useful to keep samples on ice until the denaturation step. It may also be advantageous to add protease
inhibitors and/or phosphatase inhibitors to prevent sample degradation. A good example is p53 which is
a phosphorylated protein that is highly sensitive to proteases. Without precautions the protein and
phosphorylation sites are destroyed leading to false negative results.
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After protein extraction, the concentration of protein should be measured to ensure that equal amount
is loaded onto the gel. At Boster, we have developed three protein assay kits for this purpose:
The Coomassie Plus Protein Assay is easy to carry out and consumes less sample. However, there can be
considerable variability among proteins in this assay that impacts linearity. The pH values may also
change as a function of a variety of elements such as high concentration of Tris, EDTA, urea, glycerol,
sucrose, acetone, ammonium sulfate and detergent. Further, the pH change will subsequently influence
color development and interfere with protein quantification.
The BCA protein assay is valued for increased stability, higher sensitivity and greater flexibility. The
colored complex formed is more stable with few interfering substances. Therefore, this assay has an
obvious advantage over the Coomassie Plus Protein Assay which is influenced by detergent and pH.
Electrophoretic separation describes the phenomenon of charged particles moving through a gel
matrix under the influence of an electric field. It is used to separate proteins according to their
electrophoretic mobility depending on the charge, size and structure of the molecules. Polyacrylamide
gels (PAGE) are three-dimensional mesh polymers composed of cross-linked acrylamide. As a
supporting matrix PAGE gels have several desirable features that make it a versatile medium. It is
thermo-stable, transparent, strong, and can be prepared with a wide range of average pore sizes.
Denatured Proteins
Denatured proteins are most commonly used for Western blotting. Native
protein structures may include inter- and/or intra-chain bonds creating
secondary or tertiary structures which may play a vital role in the activity of
the protein. However, these structures also affect the migration of the protein through the
pores created by the cross-linked polyacrylamide. For example, a low molecular weight
protein with a complex tertiary structure may migrate at the same rate as a larger protein with a
simpler structure indicating an apparently similar molecular weight. It is important to note
that many antibodies are generated against peptides and often better recognize protein in the
denatured state. Denaturation of proteins is accomplished in three ways:
(i) Sodium dodecyl sulfate (SDS), an anionic detergent, is used as a denaturing agent to break
hydrogen bonds within and between molecules to unfold proteins and break up secondary
and tertiary structures. The anionic (or negative) charge of SDS imparts a negative charge to
3
linearized proteins that is proportional to its peptide length. In the presence of SDS,
electrophoretic mobility is largely based on molecule weight.
(ii) Strong reducing agents such as β-mercaptoethanol (BME) and dithiothreitol (DTT) disrupt
disulfide linkages between cysteine residues within or between protein chains.
(iii) Heating the samples to 100°C further promotes protein denaturation and SDS binding, rod-
shape formation, and negative charge adherence.
Loading buffers containing tracking dyes such as bromophenol blue dye may be added to the
protein solution to follow the progress of the proteins through the gel during the electrophoretic
run.
The use of (PAGE) gels for separating proteins ranging in size from 5-2,000 kDa is
made possible by the uniform pore size created during polymerization of acrylamide.
Pore size is controlled via the ratios of acrylamide to bis- acrylamide in gel making
process. Typical PAGE gels are cast with a stacking gel (5%) poured over the top of
a resolving gel. The relatively low concentration stacking layer is designed to counteract the migration
differential of protein samples as they first enter the separation matrix. At the interface of the
two gel components, the proteins are “stacked” to optimize their separation during their migration
through the resolving gel. Resolving gels are made in 5%, 8%, 10%, 12% or 20% formulations, each
providing a range of protein separation based on apparent molecular weight. Gel concentrations are
selected based on the size of the target protein destined for analysis. Higher percentage gels are used
for separation of proteins of lower known or estimated molecular weight. If the molecular weight of
the protein is unknown or if proteins of a variety of molecular weights are examined, then a gradient
gel containing multiple percentages within the same gel can be useful. Protein samples are introduced
into the gels via wells that define the lanes of protein migration. Changes on the buffer system of the
gel can help to further resolve proteins of very small sizes.
4
Buffer Systems
A wide variety of buffer systems exist for use with PAGE. The most well-known is the tris-glycine or
"Laemmli" discontinuous buffer system which uses a stacking gel prepared with buffer of pH
6.8 and a resolving gel at a pH of ~8.3-9.0. Drawbacks associated with this system include:
potential protein deamination and alkylation or disulfide bond formation between cysteine residues
from the re-oxidation of cysteine due to non-migration of sample buffer reducing agents into the
resolving gel. Additionally, different buffers are required for the cathode and anode ends of the
gel. Evolutions in buffering technology (e.g., bis-Tris) avoid these pitfalls by casting and running
gels under slightly acidic (pH ~6.5) conditions and include reducing agents (e.g. sodium bisulfite)
that move into the gel ahead of the proteins to maintain a reducing environment. An additional
benefit of using buffers with lower pH values is that the acrylamide gel is more stable at lower pH
values, so the gels can be stored for long periods of time before use.
As voltage is applied to a Tris-glycine system the anions (and negatively charged sample molecules)
migrate toward the positive electrode (anode) in the lower chamber. The leading anion is Cl- (high
mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration).
SDS-protein particles do not migrate freely at the border between the Cl- of the gel buffer and the
Gly- of the cathode buffer. Because of the voltage drop between the Cl- and glycine buffers,
proteins are compressed (stacked) into micrometer thin layer between the stacking and resolving
gels. In resolving gel, proteins with more negative charges per unit migrate faster than those with
less negative charges per unit. That is, proteins with small molecular weight migrate faster than
proteins with large molecular weight. The boundary moves through a pore gradient and the protein
stack gradually disperses due to a frictional resistance increase of the gel matrix. For every protein at
a different position, stacking and un-stacking occur continuously in the gradient gel.
3. Protein Transfer
Transfer Types
Two main types of procedures and associated apparatus are used to perform
electrophoretic transfer of proteins. Each of them offers advantages and is selected based
upon experimental requirements and lab preferences.
Semi-dry protein transfer is a rapid, efficient and inexpensive method requiring no buffer tanks or
gel cassettes. A separation gel and transfer membrane are equilibrated in transfer buffer and
sandwiched between sheets of buffer-soaked filter paper, then clamped between two horizontal
plate electrodes. To maximize the amount of buffer available during the transfer, extra thick
filter paper is recommended. Under an electric field the negatively charged proteins migrate
toward the positive cathode, exiting the gel and depositing on the transfer membrane where they
are held by hydrophobic interactions. Transfer times for semi-dry transfers are
5
short due to both the high electric field caused by the close proximity between the two
electrodes and by the low buffer capacity of the saturated filter paper. High field strengths may
cause some small proteins to be driven through the transfer membrane, while shortened run
times may result in the inefficient transfer of some large proteins. Semi-dry protein transfer
methods are not considered to be reliable for quantitative analysis.
In a wet transfer process, the gel-membrane-filter sandwich is flanked by pads or sponges and
placed vertically in a cassette or transfer tank filled with buffer. As in the semi-dry transfer
proteins migrate from the gel to the membrane under the electric field produced by the parallel
electrodes. Large buffer capacities with wet transfer procedures offer more flexibility for
voltage settings, transfer times, and temperature conditions. Varying these parameters may
allow some larger proteins to be transferred more effectively while promoting efficient binding of
lower molecular weight proteins. While wet transfer methods require more complex
apparatus and higher buffer costs, the opportunities for optimization of transfer conditions
make the method a preferred choice when quantitation of target proteins are desired.
Transfer Membrane
Transfer membranes are selected based on their physical properties that lend them for use with
particular methods and conditions. Protein binding capacity, mechanical strength, and downstream
analysis such as re-probing or protein sequencing are considerations when choosing a blotting
membrane.
(i) Nitrocellulose
Nitrocellulose, one of the first materials used for transfer membranes, is still considered a good
choice for general purpose western blotting. Nitrocellulose is inexpensive, has a high affinity for
proteins, is easily prepared for blotting, and can be used with a variety of detection systems. Due
to its brittle nature nitrocellulose is not a preferred material for blots intended for workflows
requiring repeated handling or manipulations. Some manufacturers offer nitrocellulose
membranes with a synthetic backing giving increased strength while retaining the desirable
characteristics of protein binding and ease of wetting. When used as a transfer membrane
Nitrocellulose requires the use of methanol in the transfer buffer to remove SDS bound to
proteins, thus enabling hydrophobic interaction of the protein with the transfer membrane.
Methanol also has a shrinkage effect on the separation gel, reducing the size of pores in the
gel matrix. As smaller pore size may retard movement of some larger proteins, consideration
must be given to the gel concentration when selecting transfer membranes and protocols.
6
(ii) Polyvinyl Difluoride (PVDF)
Membranes made of PVDF have some distinct advantages over nitrocellulose such as higher
binding capacity (170-200 µg/cm2 vs. 80-100 µg/cm2) and durability. The higher binding
capacity is advantageous when attempting to detect low expression proteins, but may also lead to
higher background noise during detection. Prior to use, PDVF membranes must be
thoroughly wet in 100% methanol and then equilibrated in transfer buffer by soaking for at
least 5 minutes. Advantages of PVDF membranes are the opportunities for re-probing the
transferred proteins after a stripping procedure. WB Stripping Buffer (AR0153) can be used to
remove bound primary and secondary antibodies. Quantitative analysis of detected proteins on
re-probed gels should be avoided as the stripping process may remove some target proteins from
the membrane.
Blotting membranes are available at typical pore sizes of 0.2 µm, and 0.45 µm. For proteins of 20
kDa or smaller, 0.2 µm pore size is recommended, and 0.45 µm pore if your protein is greater than
20 kDa. If your application includes quantitation of your target protein, or your protein
concentration is low, the general rule is to use the membrane with the smaller pore size.
Membrane Formats
(i) Pre-cut membranes are available for all of the most commonly used sizes of separation gels.
Pre-cut and packaged by manufacturers, these membranes may provide more consistent
protein transfers than membranes cut from rolls or large sheets.
(ii) Rolls of membranes are available for cutting membranes yourself in your laboratory to use with
a variety of gel sizes. The lower cost of the membrane may be offset by the time expended
to ensure the size of the transfer membrane exactly matches that of the separation gel to avoid
introducing variability in transfer analysis.
4. Membrane Blocking
Following protein transfer, it is important to block the unreacted sites on the membrane using inert
proteins and/or nonionic detergent to reduce levels of nonspecific protein binding during the assay.
Blocking buffers should block all unreacted sites without disrupting target protein-membrane
interactions or affect epitope availability The most typical blocking solutions are nonfat dry milk,
casein, gelatin, or Tween-20 in TBS and/or PBS buffers.
Nonfat dry milk is the most economic choice, but should be avoided for blots using biotin-conjugated
antibodies due to intrinsic amounts of glycoprotein and biotin present in milk. Additionally, naturally
present phosphatases may lead to protein dephosphorylation, interfering with target identification of
7
phosphorylated proteins. BSA or casein in TBS are recommended for phosphorylated target analysis or
when using alkaline phosphatase-based detection methods.
Note: Use of NaN3 as a preservative in blocking reagents should be avoided when using peroxidase
detection systems due to its oxidase inhibiting properties.
After blocking the transfer membrane, a primary antibody specific to a binding site on
the target protein is diluted as appropriate and incubated with the membrane. This
procedure is typically carried out in a dish or tray of dimensions allowing the membrane
to lay horizontal while immersed in buffer. Incubation is performed overnight at 4ºC or 1-2 hours at
RT with mild agitation.
The choice of a primary antibody depends on the antigen to be detected. Both polyclonal and
monoclonal antibodies work well for western blot analysis. Monoclonal antibodies recognize single
specific antigenic epitope. The higher specificity may result in lower background on analysis. Target
epitopes that are blocked or destroyed may result in poor blot results when monoclonal antibodies are
used as probes. Polyclonal antibodies may recognize more epitopes on the target and they often have
higher affinity providing results for analysis when specific epitopes are not known or their identification is
not required for the application. Polyclonal antibodies are generally found at lower cost than
monoclonal antibodies.
After washing the membrane to remove unbound primary antibody, the membrane is
exposed to an enzyme conjugated secondary antibody. The secondary antibody is highly
specific for a site on the primary antibody and is engineered bind tightly. The most
popular secondary antibodies are anti-mouse and anti-rabbit immunoglobulins (Ig) as these animals are
commonly used as host species for production of primary antibodies. In turn, goats are used to raise
anti-mouse and anti-rabbit polyclonal antibodies. Thus, goat anti-mouse and goat anti-rabbit
immunoglobulins are the most commonly used secondary antibodies. The choice of secondary
antibody depends upon the species of animal in which the primary antibody was raised. For example, if
the primary antibody is a mouse monoclonal antibody, the secondary antibody must be anti-mouse. If
the primary antibody is a rabbit polyclonal antibody, the secondary antibody must be anti-rabbit. The
secondary antibody must also recognize the isotype of the primary antibody (IgG1, IgM, etc.).
7. Signal Detection
An enzyme bound to a secondary antibody reacts with a substrate introduced during the protein
detection stage of the western blot to generate a colored precipitate bound to the transfer membrane
resulting in visible protein bands. The target protein levels in cells or tissues are evaluated though
densitometry and location of the visible protein bands.
Chromogenic (Colorimetric Development)
8
Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes that are used
extensively. As a result of alkaline phosphatase (AP) catalysis, the colorless substrate BCIP will be
converted to a blue product. In the presence of H2O2, 3-amino-9- ethyl carbazole and 4-chlorine
naphthol will be oxidized into brown and blue products, respectively. The figure below shows two
WB results by using Boster’s DAB chromogenic detection system.
Chemiluminescence
Enhanced chemiluminescence is another method that employs HRP detection. Using HRP as the
enzyme label, the luminescent substrate (Luminol) will be oxidized by H2O2 and will produce light
(luminesce). Luminiscence enhancers combined with this substrate will induce a 1000-fold increase in
light intensity. The light is then detected and captured on photographic film. Film can be
exposed for multiple time points to optimize the signal to noise ratio. The figure on the next page
shows three WB results by using Boster’s ECL detection system.
9
Whole Cell Lysates Whole Cell Lysates Tissue Lysates
Lane 1: MCF7 Lane 1: MM453 Lane 1: Rat Ovary
Lane 2: Hela Lane 2: MM231 Lane 2: Human placenta
Lane 3: Jurkat Lane 3: Hela
Lane 4: HT1080 Lane 4: HT1080
Lane 5: Colo320 Lane 5: Colo320
Fluorescence
The choice of WB signal detection methods depends on the enzyme or detection agent bound to the
secondary antibody. ECL (Enhanced Chemiluminescent) and DAB chromogenic detection systems are
typically used. In general, chromogenic detection is not as popular as chemiluminescent due to its
decreased sensitivity.
8. Controls
Proper control design is essential to successful western blot analysis. It will guarantee accurate and
specific test results identifying various problems quickly and precisely. We will discuss five control
types as follows:
Positive Control
A lysate from a cell line or tissue sample known to express your target protein. Positive controls are
designed to verify working efficiency of the antibodies.
11
Negative Control
A lysate from a cell line or tissue sample known not to express the protein you are detecting.
Negative controls are used is to check antibody specificity. Nonspecific binding and false positive
result may be identified using negative controls.
Blank Control
Primary and secondary antibodies are not incubated with the membrane. This analysis is performed to
verify the transfer membrane itself does not induce a false positive result and is also used to check
membrane blocking efficiency.
Loading Control
Loading controls are used to check sample quality and the performance of the secondary antibody
system. Loading controls are antibodies to "house-keeping proteins" or proteins that are expressed at
equivalent levels in almost all tissues and cells. Loading controls are required to check that
equivalent quantities of target protein have been loaded across gel lanes, especially when a
comparison must be made between the expression levels of a protein in different samples. They are
also useful to check for effective transfer from the gel to the membrane across the entire gel. Where
uneven quantities of sample are loaded or even transfers have not occurred, the loading control
bands can be used to quantify the protein amounts in each lane. For publication-quality work, use
of a loading control is absolutely essential. Come commonly used loading controls are:
12
WB Result by Beta-Actin Loading WB Result by GAPDH Loading Control
Control and Enhanced and Enhanced Chemiluminescent (ECL)
Chemiluminescent (ECL) Substrate Substrate from Boster
from Boster
13
Recommended Protocol
1. Sample Preparation
A. Protein Extraction
14
varied depending on the cell type.
Discard lipid (at top) and cell debris (at bottom) by aliquotting the solution in the middle
to a fresh tube and keeping the tube at -20°C.
The test tube protocol for our BCA (Bicinchoninic Acid) Protein Assay Kit (Boster Catalog#
AR0146) is described here. Refer to our datasheet for the microplate protocol as appropriate.
Tube Diluent Volume (µL) BSA Volume (µL) BSA Concentration (µg/mL)
A 0 600 (From Tube A) 2000
B 100 300 (From Tube A) 1500
C 300 300 (From Tube A) 1000
D 200 200 (From Tube B) 750
E 300 300 (From Tube C) 500
F 300 300 (From Tube E) 250
G 300 300 (From Tube F) 125
H 400 100 (From Tube G) 25
I (Blank) 300 0 0
15
(ii) Quantitation Assay (For the test-tube protocol, ratio of sample to working range is 1:20).
Pipette 0.1 mL of each standard and unknown sample replicate into an appropriately
labeled test tube.
Add 2.0 mL of the working range (WR) to each tube and mix well.
Cover and incubate tubes with one of the protocols:
- Standard Protocol: 37°C for 30 min (WR: 25 to 2,000 μg/mL).
- RT Protocol: Room temperature for 2 hrs (WR: 25 to 2,000 μg/mL).
- Enhanced Protocol: 60°C for 30 min (WR: 5 to 250 μg/mL).
Note: Increasing the incubation time and temperature can increase the net 562 nm
absorbance for each test and decrease both the minimum detection level of the reagent and
the working range of the protocol. Use a water bath to heat the tubes for either Standard
(37°C incubation) or Enhanced (60°C incubation) Protocol. Using a forced-air incubator
can introduce significant error in color development because of uneven heat transfer.
Note: Because the BCA Assay does not reach a true end point, color development will
continue even after cooling to room temperature. However, because the rate of color
development is low at room temperature, no significant error will be introduced if the 562
nm absorbance measurements of all tubes are made within 10 min of each other.
Subtract the average 562 nm absorbance measurement of the blank standard replicates
from the 562 nm absorbance measurement of all other individual standard and unknown
sample replicates.
Prepare a standard curve by plotting the average blank-corrected 562 nm measurement for
each BSA standard vs. its concentration in μg/mL. Use the standard curve to determine
the protein concentration of each unknown sample.
2. Electrophoresis
The first step of gel preparation is to determine the gel percentage based on the molecular weight
of your protein sample:
If you are not sure of the size of your protein or are looking at proteins of a variety of molecular
weights, then a gradient gel may provide the best resolution.
16
Notes
We recommend using the SDS-PAGE Gel Preparation Kit is available from us (Boster Catalog
# AR0138). It contains most of the reagents for the gel preparation and can be used to make
both SDS-PAGE gel and native PAGE gel, respectively.
Many protocols are available for gel preparation. Please refer to the manufacturer’s guidelines
for use of specific products.
Pre-cast gels may also be used instead of making your own gel.
Mix the extracted protein sample with 4X Dual Color Protein Loading Buffer (Boster Catalog
# AR1142) at 3:1 ratio (i.e., add 300 µg sample to 100 µL Loading Buffer).
Dual Color Protein Loading Buffer is designed to prevent protein degradation during sample heating
prior to electrophoresis and is able to work against pH changes during SDS-PAGE run (Many proteins are
sensitive to pH changes that result from temperature fluctuations of Tris buffers during electrophoresis).
It contains two tracking dyes: blue (Bromophenol Blue) for tracking the progress of electrophoresis and
pink (Pyronin Y) for monitoring of protein transfer to the membrane. Refer to the datasheet on our website
for more information.
You may also use one of the following reagents/methods instead of the Dual Color Protein
Loading Buffer:
- 2X SDS-PAGE Protein Loading Buffer (Boster Catalog # AR0131) at 1:1 ratio (i.e. add 100
µg sample to 100 µL Loading Buffer)
- 5X SDS-PAGE Protein Loading Buffer (Boster Catalog # AR1112) at 4:1 ratio (i.e. add 400
µg sample to 100 µL Loading Buffer)
- 2X SDS-PAGE Tricine Protein Loading Buffer (Boster Catalog # AR1143) at 1:1 ratio (i.e.
add 100 µg sample to 100 µL Loading Buffer) if detecting protein with MW < 10 kDa
- Laemmli 2X Buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol
17
blue, 0.125 M Tris-HCl; pH 6.8) at 1:1 ratio (i.e. add 100 µg sample to 100 µL Loading Buffer)
Denature the sample/loading buffer mixture in a 100°C water bath for 5 min (or follow the
manufacturer instructions); Alternatively, the mixture can be stored in aliquots at -20°C for
several months or at 4°C for 1-2 weeks before use.
Notes:
In a discontinuous system, the electrophoresis voltage for stacking gel is lower than that for
resolving gel to ensure that proteins are concentrated on the same level before running into the
resolving gel.
* The applied voltage should be adjusted according to the gel thickness, power supply used and
resolution desired.
After electrophoresis, we recommend using one of our gel staining solutions to determine if
the electrophoretic separation works. Refer to the datasheet(s) on our website for more
information.
Note: Stained gel cannot be used in the subsequent protein transfer procedure.
18
(ii) Wet Transfer
- Soak two filter papers in a separate container with the same transfer buffer
- Crack open the transfer cassette with a spatula and make sure to loosen the cassette
hold all the way around before carefully pulling apart the two halves (Before doing
anything with the gel, such as cutting it, pay careful attention to the location of
lane #1.).
- Cut the gel according to the size of the membrane with a razor blade and then cut
the corner on the side of the gel with lane #1.
- Immerse the gel in 1X transfer buffer for 15-30 min.
- Place the gray or black plate of the transfer cassette on a clean surface.
- Place one pre-wetted foam pad on the gray side of cassette.
- Place a moistened sheet of filter paper on the foam pad.
- Carefully peel the gel off of the remaining half of the gel cassette and place it onto
the filter paper (Moisten the gel with transfer buffer and use a serologically clean
-
19
pipette or a Falcon tube, as if it were a rolling pin, to roll air bubbles out of the
membrane).
- Place the membrane onto the gel with the corners match up (Once the membrane
contacts the gel, it should not be moved or “ghost bands” can result).
- Complete the sandwich by placing a piece of filter paper onto the membrane.
- Add the second foam pad on top of the filter paper.
- Lock the transfer cassette firmly with the white latch (Be careful not to move the
gel and filter paper sandwich and make sure that the foam pads, filter papers and
membrane are thoroughly immersed in the transfer buffer).
Notes
1. The transfer can be completed overnight at a lower voltage, for example, 10 V.
2. Transfer time and voltage should be optimized according to the gel concentration. Higher
gel concentration requires additional time.
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(D) Membrane Blocking
Rinse the blotting membrane 3X using TBS Wash Buffer (20 mM Tris, pH 7.5; 150 mM NaCl;
0.05% Tween 20) [Boster Catalog # AR0144] at room temperature for 10 min each time.
After rinsing, immerse the blotting membrane in TBS Blocking Buffer (5% non-fat dry milk in
buffer of 20 mM Tris, pH 7.5; 150 mM NaCl) [Boster Catalog AR0143] and incubate for
1.5 - 2 hours at room temperature (or overnight 4°C) with shaking. Alternatively, buffer
containing non-fat dried milk, gelatin, or BSA can be used. For use with biotin systems or
detection of phosphoproteins, non-fat dried milk is not recommended.
After blocking, the membrane is incubated with a primary antibody (that binds to the target
protein) followed by a HRP- or AP-conjugated secondary antibody.
Dilute the primary antibody with the TBS Wash Buffer (Boster Catalog # AR0144); Follow
the antibody protocol from the manufacturer for optimal dilution.
Incubate the primary antibody and the membrane at 4°C overnight or for 1-2 hours at room
temperature. For the best results, incubation time and antibody concentration may need to be
optimized.
Wash the membrane 3X with the TBS Wash Buffer for 10 min each to remove unbound
antibody.
Dilute the secondary antibody with the TBS Blocking Buffer (Boster Catalog # AR0143);
Follow the antibody protocol from the manufacturer for optimal dilution.
Incubate the secondary antibody and the membrane at 4°C overnight or 1-2 hours at room
temperature on a shaker.
Wash the membrane 3X with the TBS Wash Buffer for 10 min each to remove unbound
antibody.
In this section, we provide the protocols for the Enhanced Chemiluminescence Detection (ECL)
and colorimetric detection (DAB or BCIP/NBT) methods. Use the method that fits your
preferences and criteria.
21
† Instead of using the ECL kit which provides 1) chromogenic reagents A and B (20X
concentrated; 5 mL), 2) blocking buffer and 3) HRP-conjugated secondary antibody,
one may use one of the following standalone chromogenic reagents A and B from
Boster:
Reagent A Reagent B
Product Conc. Volume Conc. Volume Catalog #
Hypersensitive ECL Substrate 1X** 100 mL 1X** 100 mL AR1170
Hypersensitive WB Substrate 20X 5 mL 20X 5 mL CR0001-5
Hypersensitive WB Substrate 20X 10 mL 20X 10 mL CR0001-10
Hypersensitive WB Substrate 20X 25 mL 20X 25 mL CR0001-25
** Ready-to-use
Prepare the ECL substrate solution by mixing the following and use the solution
within two hours of preparation:
- 50 µL of 20X Chromogenic Reagent A (Luminol & Luminious Enhancer)
- 50 µL of 20X Chromogenic Reagent B (Peroxidase & Stabilizer)
- 1 mL of distilled water
Thoroughly cover the membrane with the substrate solution (use 1 mL of solution
for 10 cm2 of membrane).
Incubate the membrane at room temperature until bands appear (usually 1-5
min; incubation time can be estimated in dark room).
Gently blot the edge of the membrane on a piece of paper to remove excess
any substrate solution.
Put a clear preservative film or transparent glass paper over the membrane and
remove any air bubbles observed.
Develop and fix the film in a dark room immediately using our recommended WB
Developing and Fixing Kit (Boster Catalog # AR0132); Alternatively, fluorescence CCD
scan, digital imager or luminometer can be used).
22
Notes
- WB Stripping Buffer (Boster Catalog # AR0153) is recommended to remove primary
and secondary antibodies on the membrane if proteins on the membrane need to be
reused.
- Use the control protein levels to normalize the target protein levels.
Choose the correct DAB kit according to the species that the primary antibody is raised
and the desirable color:
Thoroughly cover the membrane with the substrate solution (use 1 mL of solution for
10 cm2 of membrane).
Incubate the membrane at room temperature until bands appear (usually 10-30 min);
incubation for BCIP/NBT should be done in dark.
Wash the membrane in distilled water to stop the reaction.
Observe the bands and take pictures.
23
Tips for Convincing Your PI to Purchase Pre-Cast Gels
1. Every homemade gel is unique, takes hours to prepare, and once made doesn’t last more than a few
days. Pre-cast gels are consistent, ready to run in minutes, and last for months.
2. Pre-cast gels have become ubiquitous in academic and commercial laboratories worldwide,
referenced in 1,000’s of peer-reviewed papers.
3. Gel components are toxic nerve damaging substances; exposure to the process is dangerous and
unnecessary. Don’t you care about your staff (technicians, grad students, post-docs, etc.)?
4. Laemmli? Oh right, my grandfather used to talk about his gel chemistry, back in the days. Slab gels,
wow.
5. With all due respect, the 1970’s called and would like their homemade gels back.
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Safety and Good Laboratory Practices
At Boster we care about your safety so we have included this section on good laboratory
practices. These are general guidelines. Please consult your environmental health and safety
office for rules and regulations specific to your institution.
1. Always use chemical safety glasses, nitrile gloves, fully buttoned front or back closing lab coat, and
closed toe shoes.
2. Use gloves and clean blunt-ended forceps to prevent hand contact with membrane, film or reagents.
3. All handling of chemicals should be done under a chemical fume hood; always point open test tubes
away from face to minimize inhalation.
4. Acrylamide, as a monomer, is considered toxic, carcinogenic, and may cause CNS damage. It is readily
absorbed through intact skin from aqueous solutions. However, once polymerized, the solid
polyacrylamide is considered quite safe. However, PAGE gels should still be handled with gloves under the
assumption that they may still contain unreacted monomer.
5. Keep the assay kit’s product insert and be sure it is available to testing personnel. Use the product insert
for the kit currently in use; do not use old product inserts.
6. Read the product insert before running the assay to check for changes in procedures or quality control:
a. Become familiar with the test procedure. The language used to convey the instructions is important.
Words like ‘always’, ‘shall’, ‘must’, and ‘required’ mean the instruction is regulatory and must be
performed. ‘Should’ or ‘recommend’ mean the action is not regulatory, but it is good laboratory
practice to perform those actions.
b. Study each step and perform them in the proper order.
c. Know the time required for performing the test and achieving the optimal result.
d. Be sure to have all required reagents and equipment ready before actually performing the assay.
8. Avoid ignition sources; use absorbent materials to contain any spill; dispose of waste in properly labeled
hazardous waste containers.
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Troubleshooting Guide
The following guide serves as a checklist for the possible causes and solutions with respect to some of the
most commonly encountered problems from the WB assays.
High Background
2 Aggregate secondary antibody ► Filter the secondary antibody through 0.2μm filter
formation
► Use a new secondary antibody
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9 Cross-reactivity of antibody with ► Use different blocking agent (Do not use skim milk
other proteins with biotin system
17 Contaminated equipment ► Ensure all equipment and tools are clean and no gel is
left on membrane
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Weak/No Signal
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8 Too short substrate incubation ► Lengthen substrate incubation time to five minutes
time
9 Digestion of protein on membrane ► Optimize amount of blocking agent
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18 Loss of primary antibody ► Prepare fresh antibody and store properly when not in
effectiveness use
19 Inhibition of secondary antibody ► Avoid using sodium azide together with HRP-
by sodium azide conjugated antibodies
20 Loss of effectiveness in enzyme ► Mix enzyme conjugate and substrate (no color
conjugate and substrate development when enzyme is inactive)
23 Equality or nearness in values ► Try other buffers such as CAPS buffer (pH 10.5)
between target protein’s isoelectric
point and transfer buffer’s pH ► Try low pH value buffers such as acetic acid buffer
value
24 Too high methanol concentration ► Decrease methanol concentration or use isopropyl
alcohol
Nonspecific Bands
2 Gradually accumulative differences ► Go back to original non-passaged cell line & run
of protein expression profiles due current and original cell line samples in parallel
to frequent passage of cell lines
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4 Presence of new proteins or ► Check literature for other reports (also perform a
different splice variants that share BLAST search)
similar epitopes (Possible that
same protein family is detected) ► Use cell line or tissue reported on instruction
6 Formation of multi-mer on protein ► Before running SDS-PAGE gel, boil protein for 10
target minutes to disrupt multi-mer
Diffuse Bands
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FAQs
Western blotting (WB) is a technique that separates proteins based on molecular weight. Generally,
smaller proteins migrate through the gel more quickly than larger ones. After extracting the protein of
interest and determining its quantity, WB involves gel electrophoresis which serves to separate the
protein in cell lysate or tissue homogenate samples. The lysates and homogenates have to be carefully
prepared to preserve protein integrity. The separated proteins are then transferred to a membrane
followed by a blocking step to prevent non-specific binding. The protein is then next probed with
enzyme-labeled antibodies. Finally, a substrate is added, and it reacts with the enzyme to generate a
detectable product.
The appropriate amount of protein to be loaded is a variable that must be optimized by the researcher
based on the expression level of the protein of interest in the sample being tested. This also depends on
the volume of sample that can be accommodated by the gel. For example, some larger format gels can
accommodate as much as 30 µL of sample and buffer, while others can only accommodate 15 µL. We
suggest using a range of loading amounts, for example, 10 to 50 µg of total protein per well for tissue
homogenates and cell lysates and 10 to 100 ng of total protein for purified protein samples.
4. I am having difficulty obtaining a band when detecting a recombinant protein. Why is this?
Detection of recombinant proteins presents a number of challenges that must be considered. For
example, you must ensure that the recombinant protein expressed in the sample includes the
immunogenic sequence for the antibody being used. Folding of the recombinant protein may be
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different from the endogenous native form, and this may prevent access of the antibody to the epitope.
This is particularly true for tagged proteins. We therefore suggest placing the tag at the C- or N-
terminus of the recombinant protein whenever possible to reduce the chance of encountering this
problem. We also strongly recommend an endogenous positive control as part of the experimental
design when detecting recombinant proteins in WB.
Review the antibody data sheet to see whether the antibody used has been tested under reduced and
denatured conditions exclusively. If this is the case, we recommend using reducing and denaturing
conditions.
We advise reviewing the data sheet for any recommended blocking agents because antibodies can be
very sensitive to the blocking agents used. BSA will usually yield clearer results because it contains
fewer cross-reactive proteins. However, some antibodies will work better with milk which contains a
greater variety of blocking proteins.
7. Can I incubate the primary antibody at room temperature for one hour instead of an overnight
incubation at 4°C?
We recommend beginning with your data sheet which should specify the optimized incubation
conditions. Generally, an overnight incubation at 4°C will result in more efficient and specific staining.
However, many antibodies will work just fine with a one- or two-hour incubation at room temperature.
Please review the section of Troubleshooting Guide (No/Weak Signal) in this handbook.
Please review the section of Troubleshooting Guide (High Background) in this handbook.
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c. Actual modifications to the protein such as:
i. Bands at lower-than-expected molecular weight that can be the result of protein degradation (To
prevent this, use protease inhibitors and keep the samples on ice).
ii. Protein aggregation and dimerization leading to higher-than-expected molecular weight bands (To
prevent this, boil the sample in SDS-PAGE buffer containing a denaturing agent [e.g.
dithiothreitol, β-mercaptoethanol] or extend boil time. It may also be helpful to compare the results
from reducing and non-reducing gels).
Uneven, white spots may appear on the blot as a result of air bubbles being trapped against the
membrane during transfer, causing uneven distribution of the antibody on the membrane. To avoid
this, we suggest using a falcon tube to carefully roll out any air bubbles that may have become trapped
while preparing the gel for transfer. On the other hand, black spots can be the result of the antibody
binding to the blocking agent. In this case, we suggest filtering the blocking agent to avoid this problem.
The antibody may be reacting with the MW marker. We advise leaving a blank lane between the MW
marker and the first sample.
Electrophoresis may have been done too quickly or heated up too much. We suggest using a lower
voltage and a longer time period for the migration and/or performing electrophoresis in a cold
room/on ice.
14. Why do the bands for the same protein have different sizes than I expected?
The gel may have set too quickly during casting, causing uneven acrylamide percentage between the
lanes. We recommend that you review the gel recipe. Also, try adding TEMED to the gels and add a
small amount of 0.1% SDS in water to the top of the migrating gel (The SDS keeps the gel from drying
out).
15. How can I ensure adequate transfer based on MW? How can I fix an incomplete transfer?
Incomplete transfer can be resolved by using a longer transfer time or by using a higher voltage. In
general, we recommend adding 20% methanol to the transfer buffer and transferring 70 V for 1.5
hours. Transfers can also be done overnight at low voltage (i.e., 10 V). For high molecular weight
proteins, we suggest (a) reducing the methanol in transfer buffer to 5% because larger proteins can
become stuck in the gel matrix at higher methanol concentrations; (b) using a longer transfer [e.g. 70
V for 3 hours].
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It is important to note that once the membrane has been pulled away from the gel, it is no longer
advisable to attempt to rescue an inadequate transfer. Doing so will result in ghost bands as it is nearly
impossible to place the membrane precisely on the gel as it was during the transfer.
16. What percentage acrylamide gel should I use to resolve the proteins?
The size of the pores in a gel is inversely related to the amount of acrylamide used. For example, a 4%
polyacrylamide gel has larger pores than a 12% gel. Thus, gels with lower percentages of acrylamide are
generally used for larger proteins while higher-percentage gels are used to resolve small proteins. On
the other hand, gradient gels (prepared with a low polyacrylamide percentage at the top which becomes
progressively higher toward the bottom of the gel, i.e., 4 to 20%) enable the experimenter to resolve a
range of protein sizes.
Optimal antibody concentration will give you good signal with minimum background. Obviously, to
save on costs, it is desirable to use as dilute an antibody as possible to get sufficient signal while
conserving antibody. Therefore, it is generally a good idea to conduct a titration experiment with new
antibodies, using a range of concentrations that includes the manufacturer’s suggested dilution. For
example, if the manufacturer suggests a dilution of 1:100, you might also test 1:50, 1:200, 1:500 and
1:1000. This should help you determine the ideal dilution for your conditions. For antibodies with no
suggested dilution, it is advisable to begin with titration experiments in the following ranges based on
antibody format:
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Ordering Information
High-quality and innovative products are critical for maximizing the success rate of western blot-based
protein analysis. These are the same reagents developed by our scientists and released by our QC team
when delivering to our customers, so we have the experience to support every step of your WB experiment.
Protein Extraction
Gel Stains
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Protein Transfer
Blocking
Primary Antibodies
We take pride in our 2100+ antibodies that have been validated for WB applications. Our PicobandTM
antibodies (Catalog numbers started with “PB”) are carefully developed with the best immunogens to
ensure minimal cross reactivity and maximum affinity. The table below describes some of the finest
WB primary antibodies in our broad primary antibody portfolio. See the Antibody Protein Target List
for a complete list of target proteins that we provide associated antibodies for your target of interest.
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Secondary Antibodies (Biotin-Conjugated)
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Secondary Antibodies (HRP-Conjugated): Continued
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Protein Detection (Enhanced Chemiluminescent [ECL] Kit)
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Antibody Protein Target Index (Continued)
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Antibody Protein Target Index (Continued)
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Antibody Protein Target Index (Continued)
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Antibody Protein Target Index (Continued)
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Antibody Protein Target Index (Continued)
Contact Information
Boster Biological Technology
3942 Valley Ave., Suite B, Pleasanton, CA 94566 Phone:
(888) 466-3604
Fax: (925) 485-4560
Ver. 230616
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