Introduction to

Western Blotting
Principles
Technical Guidance
Data Analysis
Troubleshooting

By Claire Moore, MSc.

Your first choice for antibodies!

Introduction to
Western Blotting
Principles

Technical Guidance

Data Analysis

Troubleshooting

By Claire Moore, MSc.

 Contents
Chapter 1 Introduction Chapter 4 Data Analysis
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Why Use a Western Blot? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Antibody Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Monoclonals vs. Polyclonals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Genetically Engineered Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . 9 Densitometers and Analysis Software . . . . . . . . . . . . . . . . . . . . 31
Epitope Tags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Western Blot Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Detecting or Characterizing Protein Expression . . . . . . . . . . . . . 31
Chapter 2 Samples, Gels, and Blotting Demonstrating Antibody Specificity . . . . . . . . . . . . . . . . . . . . . . 32
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Alternate Forms of a Protein – Prion Disease . . . . . . . . . . . . . . . 32
Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Confirmation of HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Cell Lysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Tissue Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Chapter 5 Buffers and Protocols
Purified or Semi-purified Extracts . . . . . . . . . . . . . . . . . . . . . . . . 14 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Determining Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . 14 Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Loading Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Controls and Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Cell Lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Gel Electrophoresis & Protein Transfer by Electroblotting . . . . . 40
Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Immunodetection – Indirect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Blotting Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Immunodetection – Direct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Setting Up the Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Size of the Target Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Chapter 6 Troubleshooting
Confirming the Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Unusual or Unexpected Bands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Chapter 3 Immunodetection No Bands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Faint Bands or Weak Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 High Background Signal on the Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Antibody Incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Patchy or Uneven Spots on the Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Indirect vs. Direct Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Detection with Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 References and Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Test Blots, Slot Blots, and Dot Blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

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 1

Chapter
Preface
Introduction
Since Western blotting is one of the most prevalent laboratory procedures
in use today, we have produced this handy reference guide to provide
a basic overview of the key technical considerations that go into performing
successful Western blots. Beyond basic theory, we have included some
typical experiments, as well as troubleshooting advice and time-saving
pointers to make things go more smoothly. We hope you will find this Overview
manual useful, and would greatly appreciate your feedback to Western blotting, also known as immunoblotting or protein blotting,
[email protected]. is a core technique in cell and molecular biology. In most basic terms,
For those who are not already familiar with our products, we specialize it is used to detect the presence of a specific protein in a complex mixture
in antibodies, and have been manufacturing high-quality immunological extracted from cells. The Western blotting procedure relies upon three key
reagents for over 25 years. As a result we think a lot about antibodies, not elements to accomplish this task: the separation of protein mixtures by size
just how they are created, produced and used, but also what is so special using gel electrophoresis; the efficient transfer of separated proteins to
about them. If you can imagine the difficulty of finding a single needle in a solid support; and the specific detection of a target protein by appropriately
haystack, you can begin to appreciate the task accomplished by the antibody matched antibodies. Once detected, the target protein will be visualized
as it successfully finds its corresponding antigen amongst the collection as a band on a blotting membrane, X-ray film, or an imaging system.
of other potential targets present in cellular materials.
Since Western blotting is accomplished rapidly, using simple equipment
If you are choosy about your antibodies and want to find a perfect match for and inexpensive reagents, it is one of the most common laboratory
your experiment, then please give us a try. We focus on antibody production techniques. The results achieved are also easy to interpret, unique,
in small and large scale across a broad range of research areas, and for and unambiguous. Therefore, it is routinely used on its own, or along
a variety of applications. We are so confident in our antibodies and in our with other immunoassays, in research and clinical settings. An overview
ISO-certified production facilities, that we offer a performance guarantee for of the technique is shown in the diagram below:
all the applications listed on our detailed datasheets. We are also available
to help, offering technical support from trained scientists experienced in the
use of antibodies. We can address queries that arise while carrying out your Mr (kDa)
–
1 2 3 Mr (kDa) 1 2 3
experiments, and can also assist in selecting the proper antibodies and
75 75
controls. Please contact our technical team at [email protected].
50 50
Finally, if you do find yourself without access to the antibody you are seeking, 37 37
remember that we are able to identify new specificities using the latest 25 25 target
technology with our parent company MorphoSys. Our HuCAL® recombinant 15 15 protein
antibodies are identified by screening over 45 billion potential specificities
using highly sophisticated techniques to produce new monoclonals faster +
than with traditional methods.
Please visit us at www.abdserotec.com/westernblot to learn more about our
FIGURE 1 Overview of Western Blotting. Separation of protein mixtures by electrophoresis;
Western blotting products, and to search our catalog of over 14,000 antibodies.
transfer to a blotting membrane; and detection of target protein, which only
becomes visible in the final stage as a band similar to that shown in lane 3.
Lane 1: Prestained molecular weight standards. Lanes 2&3: Protein mixtures.

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 Why Use a Western Blot? In many cases, Western blots are used in combination with other key antibody
based detection techniques, such as ELISAs or immunohistochemistry.
Western blotting is an extremely powerful technique, despite its overall
In these instances, Western blots provide confirmation of results both
simplicity, because it provides additional information not readily gathered
in research and diagnostic testing. For example, with HIV and prion disease,
from other key immunological laboratory techniques. Since proteins are
Westerns are used as a key supplemental screen since their results are less
separated by size during the gel electrophoresis stage, and then detected
ambiguous, and quicker, than other methods. Moving forward, Western blots
by a specifically directed antibody, the procedure essentially confirms the
continue to be of value in confirming results from antibody arrays, making
identity of a target protein. Furthermore, when data does not match
them suitable for use in proteomics research. Specific results and data
expectations, there may be clues as to what should be investigated to
analysis will be addressed in further detail in Chapter 4.
determine the reason. Is the band of lower or higher molecular weight than
expected? Is there a single band, or are there several bands? A smaller than
expected band could indicate that the protein has been cleaved or is degraded. Antibody Considerations
Conversely, when bands are seen at higher levels than expected, this may
One of the critical features of any successful Western blot is the highly
indicate an actual increase in mass due to glycosylation or multimer formation.
specific interaction between an antibody and an antigen. The antigen,
Alternate splicing may also cause unexpected size variations, as may the
usually a protein or peptide, is the target of the antibody. The precise point
particular combination of charged amino acids found in the protein.
of interaction is between a small region of the antigen, an epitope,
and the recognition sites found on the arms of the antibody molecule.

Mr (kDa) 1 2 3
Higher molecular weight
than expected, often due
to glycosylation or
multimer formation.
Antigen
30 Lower molecular weight
22 than expected, usually Recognition sites
due to cleavage or
degradation.
Epitope

FIGURE 2 Expected vs. Actual Molecular Weight. Lane 1: Target protein band Antibody
at the expected molecular weight. Lanes 2&3: Common alterations
that modify the molecular weight.

Western blots are in wide use across a broad range of scientific and clinical
disciplines. Their ability to clearly show the presence of a specific protein both
by size and through the binding of an antibody makes them well-suited for
evaluating levels of protein expression in cells, and for monitoring fractions
during protein purification. Likewise, they are helpful for comparing expression FIGURE 3 Antibody-Antigen Interaction
of a target protein from various tissues, or seeing how a particular protein
responds to disease or drug treatment.

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 Antibodies selected for immunodetection should be Western blot tested Comparison of Monoclonal and Polyclonal Antibodies
if possible, with attention paid to the experimental conditions recommended
by the antibody supplier. Usually, Western blot positive antibodies recognize Monoclonal Polyclonal
a short linear sequence of amino acids found within the target protein that
remains intact, or becomes visible, when the target protein is fully unraveled. Specificity for a single epitope. Varying specificities to multiple epitopes.
This is because most Western blots are carried out under denaturing and
reducing conditions which remove all higher order protein structure.
In contrast, some epitopes can be conformational, forming a three-
dimensional structural configuration of amino acids that will be lost upon
denaturation of the protein. Thus, not all antibodies work in a typical Western
blot. Since Western blot procedures allow for flexibility in choosing gel
electrophoresis and blotting conditions, it is possible to modify buffers
to retain enough higher order protein structure for detection by some
antibodies. The antibody datasheet should indicate which buffer conditions
are best-suited to the particular antibody-antigen interaction.
Identifies whether a particular region Identifies the entire target protein via binding
of a protein is present. at multiple sites. Since multiple epitopes
Monoclonals vs. Polyclonals are targeted, there is a higher likelihood
of detection of the target.
The antibodies used to detect the target protein in a Western blot will
be either monoclonal or polyclonal. Both types of antibody are typically May cross-react with other proteins that Higher background and cross-reactivity possible
created when an antigen, usually a protein or peptide, is injected into share this epitope, such as isomers or due to detection of multiple epitopes, any
an animal and its immune system responds by producing antibodies common motifs. of which may be shared by related proteins.
specifically targeted against that antigen (or more precisely to various
epitopes found on that antigen). Polyclonal antibodies consist of a mixed Usually less sensitive since only a single More sensitive because signal is amplified
antibody molecule binds to each target. through the binding of several antibodies per
pool of immunoglobulin molecules that bind to several different epitopes target.
found on a single antigen. Polyclonals are usually produced in rabbits,
donkeys, sheep, and goats, and are purified from serum. More expensive to produce initially, Less expensive to produce initially, but supply
but available in an unlimited supply. is limited to immunized animal(s). There will
In contrast, monoclonal antibodies bind to a single epitope within a target be greater variability between preparations.
antigen. They are composed of homogeneous cloned immunoglobulin
molecules, rather than the heterogeneous antibody mixture typical Genetically Engineered Antibodies
of polyclonals. Monoclonals are made by fusing antibody producing cells
from the spleen of the immunized animal (usually a rat or mouse) with In addition to traditional monoclonal and polyclonal antibodies targeted
an immortalized cell line to produce single specificity antibodies that against specific proteins, there are other means of antibody generation and
can be purified from tissue culture supernatant. Both monoclonals and protein detection available as the result of numerous advances in genetic
polyclonals are used in Western blotting, and offer various advantages engineering technology.
and disadvantages that are summarized in the accompanying table.
It is now possible to create and produce antibodies using fully in vitro techniques
such as phage display in conjunction with highly complex libraries which
represent the vast array of potential antibody binding regions. At AbD Serotec,
we routinely produce human recombinant monoclonal antibodies for research,
clinical, and diagnostic applications with our HuCAL® Technology.

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 2

Chapter
Epitope Tags
If there are no antibodies available to the protein of interest, it is still
Samples, Gels, and Blotting
possible to carry out a range of immunodetection techniques, including Western
blotting, by using epitope tags and matched epitope tag antibodies.

This elegant strategy works by adding a small sequence of DNA that codes
for a known antigenic epitope during cloning of the protein of interest.
Since matched antibodies already exist that will specifically bind to this epitope,
the target protein can be detected because it also expresses the appropriate
Overview
epitope. Therefore, immunodetection can be carried out quickly and without the A typical Western blot, or immunoblot, relies upon a purified, semi-purified,
need to wait for the generation of unique antibodies to a newly identified target or crude extract of cellular proteins containing a target protein that can
protein. This technology is also of significant benefit when working in organisms be detected by antibodies. Several key steps are required to take the sample
where few specific antibodies are readily available. One downside to this from the cellular starting point to a detectible band on a Western blot.
technology is that the target protein is altered by the addition of tag, This chapter focuses on the preparative stages that are accomplished prior
and thus it is not identical to native forms of the protein. to immunodetection by antibodies. Throughout these processes it is essential
that the cellular protein is prepared and stored carefully, since this will
There are a wide variety of epitope tag antibodies available, including: significantly impact the experimental results.
His-6 (MCA1396), V5 (MCA1360), c-myc (MCA2200), and others which are
The three key preparative stages are:
supplied by AbD Serotec. Epitope tag antibodies are available with a range
of common antibody labels allowing one to switch experimental techniques • Sample production by lysis or homogenization to solubilize
or detection systems without having to modify the target protein. In the Western and release cellular proteins.
blots shown below, myc-tagged KSR (Kinase Suppressor of Ras) is detected • Separation of protein mixtures using gel electrophoresis.
through the use of either an anti-myc antibody or an anti-KSR antibody. • Transfer of separated proteins to a blotting membrane which
A: B:
can be manipulated more easily than a gel.
Anti-KSR
A B antibody
Mr (kDa) 1 2 3 4
Mr (kDa)
–
1 2 3
160
105 KSR Myc-tag 75
75
50
50 37
Anti-myc
antibody 25
35
15

+
FIGURE 5 Sample Preparation, Electrophoresis, and Transfer
FIGURE 4 Western Blot and Model of Myc-tagged KSR. PANEL A: Myc-tagged murine
KSR expressed in COS cells is detected using either anti-KSR (MCA2106) Western blots are effective in detecting low nanogram to low picogram
in lanes 1&2, or anti-myc epitope (MCA2200) in lanes 3&4. Lanes 1&3: myc-KSR amounts of target protein, depending on the antibodies used and the detection
vector. Lanes 2&4: vector alone control. Note that an identical band is produced substrate chosen. If the target is suspected to be of very low abundance,
with both antibodies. PANEL B: KSR target protein with c-myc epitope tag. or if there is no detectible signal on the blot, then it may be necessary
KSR-myc fusion is detectible by both anti-KSR antibodies and anti-myc antibodies. to concentrate, immunoprecipitate, or fractionate the starting material.
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 Sample Preparation The amount of lysis buffer is determined based upon a cell count,
or else it is estimated based upon the size of the tissue culture vessel.
Cell Lysates The accompanying table provides some suggested starting points. Sometimes
mechanical disruption, such as with sonication or dounce homogenization,
Crude cellular lysates are the most common direct source of starting material is required to fully release proteins from certain cell and tissue samples.
used in Western blotting. They can be prepared from immortalized cell lines Sonication is also used to break down cellular DNA which can interfere either
known to express the target protein, or from transfected cells carrying a protein due to its high viscosity or via non-specific binding. After lysis and
expression vector. Many different cell types (mammalian, insect, yeast, centrifugation, the amount of protein in each lysate is measured.
or bacteria) can be used to supply the protein needed with slight variations
in the preparation procedure. In most cases, the cells are harvested, washed, Cell Lysis Volume Recommendations
and lysed to release the target protein. For best results, all these steps should
be carried out in a cold room, or on ice. This will minimize proteolysis,
Type of cells Amount of material Volume of lysis buffer
dephosphorylation, and denaturation, since all begin to occur once the cells
are disrupted. It is possible to simply lyse cells directly in gel loading buffer Tissue Culture 107 cells or 1 ml
if a quick check is all that is required. However, sonication may be neccessary 100 mm dish
to disrupt the highly viscous cellular DNA. Usually, 20-50 mg of cellular lysate
is loaded per lane for gel electrophoresis.
Whole Tissue 100 mg Add 2 ml and sonicate or
dounce homogenize
Harvest and wash Lyse cells on ice
cells on ice to release proteins
Bacteria Spin sample, estimate volume Add 10 volumes and vortex

Yeast Spin sample, estimate volume Add 10 volumes, then sonicate

or vortex with glass beads

Tissue Samples
Tissue samples display a higher degree of structure than cultured cells and
FIGURE 6 Cells are Harvested, Washed, and Lysed to Release Proteins thus may require higher levels of mechanical intervention in order to release
the protein of interest. They may also contain multiple cell types which are
Choosing the proper lysis buffer and determining an appropriate volume is often
differentially responsive to the lysis buffer chosen. Smaller solid tissue samples
a trial and error process that is affected by the type of protein being isolated
(up to 100 mg) are placed in ice cold extraction buffer and homogenized
as well as the particular cells used as a source. Lysis buffers vary from very gentle
on ice, usually with sonication or a douncing rod to facilitate cellular disruption.
ones with no detergent to harsher solutions such as RIPA (Radio Immuno
Alternatively, and more often with larger tissue samples, a blender is used
Precipitation Assay) buffer, which is denaturing and contains multiple detergents.
to homogenize the tissue in PBS, and then cell lysis buffer is added. Once the
Typically, NP-40 (Nonidet P-40) lysis buffer, with a milder non-ionic detergent,
tissue has been homogenized and lysed, the solubilized cellular components
is used for the isolation of soluble cytoplasmic proteins. At other times, RIPA
are clarified by centrifugation and tested for protein concentration prior
buffer is chosen because it reduces background, and because sometimes multiple
to loading on a gel.
detergents are required to fully release membrane bound or nuclear proteins.
Consideration should also be given to the antibody-antigen interaction which
may be affected by the changes to the target protein during lysis.

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 Purified or Semi-purified Extracts Sample Buffer Conditions
The simplest source of starting material for Western blotting is purified
or semi-purified protein samples that are produced in the course of protein Gel Conditions SDS DTT or bME Comments

purification. These samples rarely require any further manipulation and are Denaturing & + + Removes all higher order structure,
simply mixed with gel electrophoresis loading buffer (Laemmli sample buffer). Reducing including disulfide bonds
When using a purified or semi-purified protein preparation, it is possible to
Denaturing + - Higher order structure is disrupted,
load a much smaller amount of total protein onto the gel. Usually 0.5-1 mg
but disulfide bonds are retained
of purified or semi-purified protein is sufficient to observe a strong signal.
If unsure, results can be improved by loading several dilutions of the sample. Reducing - + Disulfides are removed, but most
higher order structure remains

Native - - The protein retains higher order

Determining Protein Concentration structure. Multimers and protein-
protein interactions can be detected
To ensure that samples are in the proper range of detection for the assay,
and so they can be compared on an equivalent basis, it is important to know
the concentration of total protein in each sample. There are various methods
Controls and Standards
available for determining protein concentration using in-house or commercially
supplied kits and reagents. The simplest method entails measuring the It can be very useful to include a positive and a negative control on the
absorbance of the lysate solution at 280 nm or 205 nm. Alternatively, several gel along with the samples that are being evaluated. For a positive control
protein assays are available which rely upon the reduction of metal ions by it is typical to use a known source of target protein, such as purified protein
the peptide bond, e.g. the Lowry and BCA assays; or by dye binding, as with or a control lysate, under conditions where it will be detectible by the antibody
the Bradford assay. In all instances, a color change results that is proportional used in the experiment. The positive control is important for confirming
to the amount of protein in the sample. Protein concentration is determined by the identity of the target since it will produce a reference band on the blot
comparison of the target samples to a known standard, such as BSA (Bovine showing the expected migration of the target protein and confirming the
Serum Albumin) diluted in lysis buffer. To get the most accurate measure of activity of the antibodies. Positive controls are also helpful for troubleshooting,
protein concentration, it is advisable to test a few dilutions of the sample and for comparing the data between separate blots. Premade cellular and tissue
ensuring that the results lie in the linear range of the protein assay. lysates are now commercially available from a number of suppliers, including
AbD Serotec, and can be used as a convenient positive control. In addition,
Loading Buffer commercially supplied tissue and cell lysates are suitable for troubleshooting
protocols. If possible, it is also helpful to include a negative sample control,
Once the protein concentration has been determined, samples are diluted
such as known null cell line, as a means of confirming that the signal is specific
in gel loading buffer, also called 2x Laemmli sample buffer. This buffer contains
to the desired protein.
glycerol so that the samples sink easily into the wells of the gel, and a tracking
dye (bromophenol blue) which migrates through the gel first to indicate how The final component required for the gel is a molecular weight standard since
far the separation has progressed. For most routine Western blots, SDS (sodium a key feature of Western blotting is to provide information on the size of the
dodecyl sulfate) and a reducing agent are also present in the gel loading/ protein. Also known as molecular weight markers, these are premade mixtures
sample buffer to fully denature the protein and remove all higher order of proteins with known molecular weights, usually 5-6 proteins spanning the
structure. See the accompanying table for the range of loading buffer options range from 10 kDa to 200 kDa. Molecular weight standards come in a variety
and the supplements they contain. Samples are heated in gel loading/sample of formats, including unstained, prestained, multi-colored, or directly labeled
buffer for either 5 minutes at 100°C, or 10 minutes at 70°C to aid in the for Western detection. They are an excellent means of monitoring progress while
denaturation. At this point, samples can remain at room temperature if they the gel is running, of checking transfer efficiency, and for orienting the
are to be used immediately, or placed at 4°C or -20°C for later analysis. immunoblot. Care should be taken not to overload the standards since they
may obscure signal from the target protein.
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 Gel Electrophoresis The percentage and the thickness of the gel will impact the transfer
of proteins out of the gel in the blotting phase, so using a thinner gel,
After the samples have been prepared, they are separated by size using
or a lower percentage of acrylamide, may improve transfer results.
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
Since the samples have been denatured in gel loading buffer containing Once the gel sets, it is placed into the running apparatus. Small volumes
SDS detergent, the protein is uniformly negatively charged and will now migrate of protein (5-20 ml) dissolved in gel loading buffer are added to each
in an electric field through the gel and towards the positive electrode. Since individual well. The gel is then connected to a power supply and allowed
the charge to mass ratio is equalized by the binding of SDS consistently along to run for a few hours in a buffer tank to separate the proteins. If the gel
the length of the proteins, and higher structure has been removed, the proteins is run at too high a voltage it will overheat and distort the bands.
will be separated primarily by size. The key is to effect a separation such that The accompanying gel shows cellular lysates which have been well-separated
the target protein will be properly resolved from the other components of the on a gradient gel, and stained with Coomassie dye to visualize all the
mixture. This makes it possible to clearly identify the target protein later through separated protein bands.
immunodetection with a specific antibody.

Most often the gel is made and run under denaturing conditions (25 mM Tris Mr (kDa)
base, 192 mM glycine, 0.1% SDS, at pH 8.3). However, alternative gel conditions Mr (kDa)
can be used, depending upon the protein under investigation and the aims of
260
260
the experiment. Options include: non-denaturing, non-reducing, native, two- 160
160
dimensional separations (by size and by isoelectric point), and buffer variations 110
110
80
80
that are more suited to the separation of smaller or larger proteins. It is 60
60
important to make sure that the buffer used is compatible with later steps in 50
50
the procedure and the blotting materials. 40
40
30
30
SDS-PAGE gels (commercially supplied or made in-house) usually consist
of a main gel, which is poured between two glass or plastic plates, and which 20
20

is sometimes topped by a short stacking gel. Gels can be made with a uniform 15
15

acrylamide percentage, or with a continuously varying gradient that yields 10

10
improved resolution over a broader range of molecular weights. See the table
below for some common gel percentages and their separation ranges. 3.5
3.5

Polyacrylamide Gel Percentage Separation Ranges

Acrylamide Concentration (%) Separation Range (kDa) FIGURE 7 HeLa Cell Lysates (LYS001) Separated on a 4-12% Gradient Gel

5 60-210

7.5 35-95

10 15-70

15 4-45

4-12 gradient 5-200

4-20 gradient 4-200

10-20 gradient 3.5-110

16 C hapte r 2 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 2 17

 Blotting
Following gel electrophoresis, the separated protein mixtures are transferred
–
to a solid support for further analysis. While it is possible to use diffusion +
or vacuum assisted transfer, electroblotting (Towbin et al.,1979) is the method
relied upon in most laboratories, due to the speed and efficiency
of transfer. The electric field used for the transfer is oriented perpendicular
to the surface of the gel causing proteins to move out of the gel and onto Fiber pad
the blotting membrane, which sits between the gel surface and the positive
electrode. The gel and blotting membrane are assembled into a sandwich along
with several sheets of filter paper which protect the gel and blotting membrane
and help to ensure close contact between their surfaces. It is imperative that
the membrane is placed between the gel and the positive electrode so that
the negatively charged proteins migrate from the gel onto the membrane.
The transfer buffer used for electroblotting is similar to gel running buffer
with the addition of methanol which helps proteins bind to the blot.
+
Electrophoretic transfer can be accomplished under wet or semi-dry conditions.
Filter papers
In a wet transfer, the gel/blotting paper/filter paper sandwich is placed into
a cassette along with protective fiber pads. The cassette is then immersed Wet transfer
in a buffer tank and subjected to an electrical field. With semi-dry transfer, Blotting membrane
the gel/blotting paper/filter paper sandwich is assembled on large electrode Proteins
plates which generate the electric field, and buffer is confined to the stack move Gel
of wet filter papers.
Semi-dry transfer
Transfer times vary from 1 hour (semi-dry transfer) to several hours or overnight Filter papers
(wet transfer). Wet transfer is usually considered to be more reliable because it is
less likely to dry out the gel, and is often preferred for larger proteins. Since there –
will be significant variation in the chosen transfer system, it is best to consult the
manufacturer of the equipment used for specific instructions.

Blotting Membranes
The solid support onto which the separated proteins are transferred is usually
of two types, nitrocellulose or polyvinylidene fluoride (PVDF) membrane, both
of which bind proteins with high affinity. Nitrocellulose has been in use for
a long time, and is sometimes preferred because of its excellent protein binding
and retention capabilities. However, nitrocellulose is brittle and thus it is usually
less effective when blots need to be reused. PVDF demonstrates superior
mechanical strength making it suitable for stripping/reprobing and for further
protein characterization techniques, such as sequencing and proteolysis
Sometimes higher background staining is seen with PVDF membranes, FIGURE 8 Western Transfer Methods
and extra care must be taken to prevent this from occurring.

18 C hapte r 2 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 2 19

 Setting Up the Transfer of prestained markers on the blot as compared to the gel. However,
this is only a crude measure since it provides results for a single lane.
While the gel equilibrates in transfer buffer, the blotting membrane
A more reliable method of confirming transfer is through the use of
is prepared. Often the blotting membrane is supplied precut to size, or else
a reversible stain which identifies the presence of protein bands directly
it is precisely cut to match the size of the gel. Since oils present on the hands
on the membrane. Ponceau S, a red stain applied in an acidic solution,
will interfere with signal on the blotting membrane, it is advisable to wear
is typically relied upon for this purpose because it is compatible with all
gloves while handling the blotting membrane. Usually a corner is marked
types of immunodetection labels and substrates. The procedure is very simple
for later orientation, though this is not necessary when using pre-stained
to use and yields results in about 10 minutes. When unlabeled molecular
molecular weight markers in one of the side lanes. Prior to assembly in the
standards are used, it is necessary to mark the position of the molecular
transfer apparatus, the blotting membrane is prewetted. Nitrocellulose is
weight standards directly on the blot when they become visible with
prewetted by floating and then immersing in dH20 or transfer buffer, and
the Ponceau S dye since they will be needed for reference at a later point.
PVDF is prewetted in methanol. Both types of membrane are then soaked
in transfer buffer and applied directly to the surface of the gel. The only negative aspect of Ponceau S staining is that it doesn’t always
produce a good photographic record because the bands can be diffuse.
It is extremely important that no air bubbles are allowed to remain between
In the accompanying stained blot, it is possible to see differing quantities
the gel surface and the blotting membrane, since air bubbles will disrupt the
of cell lysates that have been transferred along with multi-colored molecular
transfer of any proteins in that area distorting the results. Air bubbles can be
weight markers at the far left.
easily removed by pushing gently across the surface with a spatula or rolling
with a pipet. It is wise to double check the orientation of the gel and blotting Mr (kDa)
membrane with regard to the positive electrode so that the proteins migrate Mr (kDa)
260
260
in the proper direction.
160
160
110
110
Size of the Target Protein 80
80

60
60
The size of the target protein should be considered when choosing transfer
conditions. Generally, smaller proteins will transfer out of the gel faster, 50
50
40
40
and may actually transfer through the blotting membrane into the filter papers
beyond. Blotting membranes with a smaller pore size can be used for small 30
30
proteins and peptides, and SDS can be reduced or eliminated from the transfer
buffer to improve binding to the membrane. If there is a suspicion that the 20
20

protein is transferring through the membrane, then a second membrane 15

15
can be included behind the first to catch proteins that migrate through.
10
10

In contrast, large proteins can be slower to elute and may be retained

within the gel, so overnight wet transfer is usually preferred. Bolt et al. FIGURE 9 Ponceau S Stained Western Blot of HeLa Cell Lysates (LYS001)
have developed buffer conditions which improve transfer efficiency across
a range of protein sizes, including very large proteins. There are several alternative methods that can be used in place of Ponceau S
staining. One option is to use a metal ion stain, such as copper, or metal chelate
stain. Another option is to stain the blot with India ink, which is very sensitive,
Confirming the Transfer
inexpensive, and reliable. However, since India ink creates a permanent record
Once the blotting step has been completed, the apparatus is carefully it will obscure the signal from colorimetric detection reagents. Coomassie Blue
disassembled and the success of the transfer is evaluated. The simplest stain is not typically used directly on a Western blot prior to immunodetection;
method of confirming the transfer involves noting the appearance instead, it is sometimes used to stain the gel after transfer in order to identify
what remains behind.
20 C hapte r 2 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 2 21
 Immunodetection
3

Chapter
Blocking
Blocking is a very important step in the immunodetection phase of Western
blotting because it prevents non-specific binding of antibody to the blotting
membrane. The most commonly used blocking solutions contain 3-5% BSA
or non-fat dried milk (also known as Blotto or BLOTTO) in a solution of PBS
(phosphate buffered saline) or TBS (tris buffered saline). Often, a small
Overview amount of Tween®20 detergent is added to blocking and washing solutions
to reduce background staining, and the buffer is known as PBST or TBST.
After blotting, the target protein will be detected using appropriately matched When choosing between these buffers, it is important to note that TBS/TBST
and labeled antibodies. The typical immunodetection stage involves a few is preferred with AP (Alkaline Phosphatase) labeled antibodies because PBS
basic steps: will interfere with the AP signal.
• Blocking - The blot containing the transferred protein bands is incubated Non-fat dried milk is considered to be a good starting point when selecting
with a protein or detergent solution which covers the entire surface a blocking solution because it is inexpensive and in very wide use. However,
so that antibodies do not bind non-specifically to the membrane. milk proteins are not compatible with all detection labels, so care must be
• Antibody incubation - Labeled antibody binds to the target protein taken to choose the appropriate blocking solution for the antibodies, buffers,
band present on the blot in a one-step or two-step procedure. and detection reagents. For example, BSA blocking solutions are preferred
with biotin and AP antibody labels, and anti-phosphoprotein antibodies.
• Detection with substrate - The label attached to the antibody, This is because milk solutions contain casein, which is itself a phosphoprotein,
usually an enzyme such as HRP (Horseradish Peroxidase), is detected and biotin, thus it will interfere with the assay results. Commercially supplied
using a substrate which produces a visible signal corresponding blocking solutions, such as Block Ace (BUF029) from AbD Serotec, are very
to the position of the target protein. convenient to use and can improve consistency of results, especially when
non-specific background signal is an issue. After blocking, the blot is rinsed
Mr (kDa) 1 2 3 in wash buffer, usually TBST, with gentle agitation and in sufficient volume
Add Add to keep the blot submerged. Please refer to Chapter 5 for detailed protocols.
antibodies substrate 75
50 Antibody Incubation
37
After blocking and washing, the blot will be incubated in a dilute solution
25 target
of antibody, usually for a few hours at room temperature or overnight
15 protein
at 4°C. The antibody is diluted in wash buffer (PBST or TBST) or a diluted
blocking solution, the choice depends upon the antibody. At AbD Serotec,
we offer a HISPEC assay diluent (BUF049A) which can be used with primary
and or secondary antibodies to reduce cross-reactivity and minimize
FIGURE 10 Immunodetection Overview. The Western blot is blocked, incubated with non-specific binding. Since antibody preparations vary in their levels of
antibodies, and treated with substrate to make the target protein visible. Wash purity and specific binding properties, there will be differences in the level
steps are carried out between incubations to remove excess unbound material of dilution required. For example, purified antibodies are usually diluted
and to minimize non-specific signal on the immunoblot. to a 1-10 mg/ml final concentration. The manufacturer’s datasheet should
provide dilution recommendations for a particular preparation. However,
it is best to test a range of dilutions with each new antibody, optimizing
conditions for the samples under consideration. Dot blots, slot blots,
or test blots (see the end of this chapter) can be used for checking various

22 C hapte r 3 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 3 23

 antibody concentrations. It is critical that all immunodetection steps (blocking, Comparison of Direct and Indirect Detection Methods
antibody incubation, substrate incubation, and all intervening washes) have
a sufficient volume and gentle agitation to keep the blot evenly exposed to the
Direct Detection Indirect Detection
reagents without drying throughout the process.
Advantages Advantages

Indirect vs. Direct Detection Faster overall, since there are fewer steps. Often gives a stronger signal because
multiple secondary antibodies bind
Antibody detection of the target protein is accomplished using a one-step Less chance of non-specific signal. to each primary antibody.
or two-step protocol. The one-step procedure, direct detection, relies upon
a single antibody which has been covalently joined to an easily detected label Easy to change label type or detection
molecule (biotin, an enzyme, or a fluorescent dye). Labeled primary antibodies methods for a new experiment
can be ordered directly from AbD Serotec or other commercial antibody suppliers. by swapping secondaries.
In addition, it is possible to directly label an antibody by using a commercially Saves labeling time and expense, especially
supplied labeling kit such as our LYNX Rapid Conjugation Kits®, or with when all primary antibodies are made in the
in-house reagents. same species.

With indirect detection, two different antibodies are used in sequence Provides access to a wider range of labels.
for the detection step. First, the Western blot is incubated with an unlabeled
primary antibody directed against the target protein. After washing,
a labeled secondary antibody is used to detect the presence of the primary Disadvantages Disadvantages
antibody, and thus the target protein. The labeled secondary antibody is typically
 oupling of label to the primary antibody may
C More non-specific signal can arise from
directed against the immunoglobulin class or subclass of the primary antibody’s affect the antibody’s ability to bind to the the binding of the secondary antibody
species. For example, one of our popular secondaries is STAR88P, a purified target protein. to other proteins on the blot.
donkey antibody raised against goat/sheep immunoglobulin (IgG) which
is coupled to an HRP label. Biotinylated primary antibodies also require L abeling every primary antibody adds Extra incubation and wash steps add time
time and cost. to the experiment.
a two-step detection procedure; however, the second step involves incubation
with streptavidin, a bacterial protein, conjugated to HRP (or AP), rather than with
a labeled antibody.

Detection with Substrate

Now that the target protein has been specifically tagged with an appropriately
labeled antibody, and excess antibody has been washed away, the label will
be used to identify the location of the target protein on the blot. Some labels
can be detected immediately, without any further processing. Fluorescent tags,
for example, simply require the right equipment to observe and record the
fluorescent signal. In Figure 12, a Western blot is probed with an anti-tubulin
primary antibody and a DyLight® 800 (STAR117D800) secondary antibody.
It is then detected using the LI-COR® Odyssey Infrared Imaging System. Note
Direct detection uses a Indirect detection uses an
here that the molecular weight standards at the far left are labeled to produce
labeled primary antibody to unlabeled primary followed by
a red signal allowing them to be easily distinguished from the samples. This
identify the target protein. labeled secondary antibodies.
highlights one of the advantages of fluorescent labels, which is that multiple
FIGURE 11 Direct vs. Indirect Detection labels can be seen on the same blot in the same experiment.
24 C hapte r 3 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 3 25
 Mr (kDa)
Colorimetric substrates for HRP produce brown (DAB) or purple/black (4CN,
TMB, NBT/BCIP) bands directly on the surface of the blot. These substrates are
very easy to use and take from a few minutes to a few hours to produce
85 visible bands. Detection limits for colorimetric substrates are in the low
54 nanogram range. The colorimetric detection reaction proceeds until stopped,
leading to the risk of overdevelopment and less flexibility of results produced.
29 The signal also tends to fade over time, so the record is less permanent.
20 More routinely, HRP is used with ECL (enhanced chemiluminescence)
detection. For ECL detection, the substrate is luminol which is oxidized by
HRP in the presence of H2O2 and an enhancer to produce light. The emitted
FIGURE 12 Western Detection of Tubulin using STAR117D800 light is detected by exposing the Western blot to X-ray film, or by using
a CCD camera for light capture. The emitted light forms a band on the film,
The most common antibody label used in Western blots is HRP, a small, or on the screen of the imaging system, indicating where the HRP-labeled
stable enzyme with high specificity and rapid turnover. HRP is deactivated antibody has bound to the target protein.
by sodium azide, so it is imperative that no azide is present in the blocking,
dilution, or washing solutions. At AbD Serotec we offer a specialized buffer ECL detection of HRP is extraordinarily sensitive, allowing for the visualization
for the long term storage and dilution of HRP labeled antibodies, BUF052A, of picogram to femtogram amounts of target protein. Furthermore, since
which can be used with all antibodies except those generated against rabbit multiple film or CCD exposures can be made with ease, little effort is required
immunoglobulins. The HRP label is detected when it is exposed to a substrate to produce a suitable permanent record for analysis or publication. Detection
solution in the final step of the immunodetection procedure. Substrate times for ECL substrates range from a few seconds to about 30 minutes and
solutions for Western blotting are chemical reagents that are acted upon can usually be done or redone over a period of several hours. Some molecular
by the enzyme to yield a signal that can be easily measured. HRP label is weight markers are designed to produce a signal during ECL detection
typically detected with either colorimetric or chemiluminescent substrates. yielding a visible ladder that is convenient for identifying the bands produced
during immunodetection. A discussion of Western blotting results is found in
the next chapter.
Detectible
signal Test Blots, Slot Blots, and Dot Blots
Test blots, as their name implies, are very simple Western blots that are
Substrate created for the express purpose of optimizing or troubleshooting experimental
conditions. They are usually produced by running multiple lanes of the same
lysate or purified protein solution on a gel, and after transfer cutting the blot
into strips to be tested individually. They provide a quick and efficient means
of examining a range of antibody dilutions or detection substrates.

Dot blots and slot blots are also a very useful variation on the typical Western
blot. They do not require gel electrophoresis, so there is no separation
of proteins by size. Instead, the target protein or cell lysate mixture is added
FIGURE 13 Detection of the Antibody Label. The antibody label is exposed to substrate directly onto the surface of the nitrocellulose or PVDF membrane. Protein
in the final stages, creating a visible band either on the surface of the blot solutions can be applied directly in a small volume, or with a vacuum
(colorimetric substrates), or as light emission (ECL substrate) captured manifold to produce an orderly grid of samples similar to that seen in Figure
on X-ray film or with a CCD camera. 14. Each dot or slot blot would contain known amounts of target protein

26 C hapte r 3 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 3 27

 Data Analysis
4

Chapter
or cell lysate. Once dry, dot blots and slot blots are subjected to the same
immunodetection steps used for Western blotting, i.e. blocking, antibody
incubation, and target detection with substrate. Grey and black spots on the
figure below indicate which samples are positive for the target protein and
correspond roughly to the bands produced on a Western blot.

Overview
The data produced with a Western blot is usually quite easy to interpret.
In the majority of cases, bands corresponding to the target protein will
become visible upon treatment of the blot with substrate. Their identity
is confirmed by comparison to molecular weight markers (for size)
and a positive control (size and signal). In some cases the data may
1. Block
be more complex, showing unexpected sizes, multiple bands,
2. Antibody Incubation
3. Target Detection or alteration in bands following a particular treatment. The focus of this
chapter is analysis and as such the data itself will be discussed along
with examples of different types of Western blot data from research
and clinical settings to demonstrate the flexibility of the technique.
If there are no bands on the blot, or if there are unexplained blotches
or uneven signal, troubleshooting advice is offered in Chapter 6.

Data

FIGURE 14 Dot Blots and Slot Blots. The dot blots at the left represent a dilution series Quantitation
of a sample, with smaller lighter dots corresponding to lower concentrations It is very important to be aware that the data produced with a Western
of target protein. The slot blots represent a group of random samples, blot is typically considered to be semi-quantitative. That is, it provides
the intensity of the signal corresponds to the concentration of the target a relative comparison of protein levels, but not an absolute measure
protein in that sample. of quantity for a specific target protein in a particular experiment.
The reason for this is two-fold: first, there will be variations in loading
By making a number of identical dots or slots of a single known protein
and transfer rates between the samples in separate lanes and on
sample, or a range of sample dilutions, one can quickly test several
separate blots that will need to be normalized before a more precise
combinations and concentrations of primary and secondary antibodies.
comparison can be made; second, the signal generated will not be
Dot blots and slot blots are also beneficial when screening a large number
linear across the concentration range of samples due to substrate
of samples, or if a simple answer will suffice. The main downside to slot
availability and linear responsiveness of the detection method. Since
blots/dot blots is that they provide no information about molecular weight.
the signal produced is not linear, it should not be used to attach
Thus, it is harder to detect false positive signals, or to tell whether modified
a precise concentration to a particular sample. ELISAs are more
forms of a protein are present.
suitable for this purpose and generally more sensitive.

28 C hapte r 3 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 4 29

 Normalization ECL signal can also be detected with CCD cameras, which usually have a better
linear range than film and associated analysis software. Consult the manual for
In order to compare target protein expression levels between several different the densitometer or imaging system in use for specific instructions.
samples on the same blot or across blots, it is necessary to use a loading
control to normalize the data. Loading controls are not actually anything
that is loaded onto the gel; instead, they refer to a means of equalizing Western Blot Examples
differences in gel loading and transfer rates between samples. They are not
required for every gel that is run, but are necessary for publication quality work,
and when the actual signal level between samples is compared. Typically,
Detecting or Characterizing Protein Expression
the blot is probed with an antibody to a well-characterized housekeeping gene One simple and common use for Western blotting is to identify whether
which is used as a general measure of protein expression levels in the cells used a particular protein is present or absent in a sample by looking for a band
as a source for a particular sample. Probing with the housekeeping gene of the correct size on a Western blot. This routine type of Western blot
antibody can be carried out along with the target antibody, separately is used to either test for endogenous cellular expression of a target protein,
by cutting a blot between the expected band locations, or later after the blot or to examine transfected cell lines to see if expression has been conferred
has been stripped of previously bound antibodies. Common loading controls by the introduction of a DNA construct. It is also used with techniques such
measure the levels of GAPDH, beta actin, tubulin, and histones. These proteins as protein purification and cellular fractionation to identify which samples have
vary in molecular weight and should be carefully chosen depending upon the target protein, and thus is an aid to deciding which samples to combine
the target protein and experimental conditions since there can be some or discard. After immunodetection, a band corresponding to the size of the
variation in their signal. In the Western blot below, Lnk (lymphocyte adaptor target protein should become visible in positive test samples and the positive
protein) expression is compared to non-infected cells and an unrelated protein control, in comparison to a known null cellular sample. In the figure below,
as a negative control. The matched anti-tubulin probed section of the blot HEK293 cells transfected with three isoforms of the Homer 1 protein and are
demonstrates that an equivalent amount of sample was loaded in each lane. compared to rat cortical extract as a positive control. The blot shows that the
antibody used, AbD Serotec’s AHP737, recognizes Homer isoform 1c, but not 2b
1 2 3 4 5 or 1a. The presence of untransfected cells (Lane 2) confirm that the signal is
specific to transfection with the appropriate construct. The faint band seen in
68 kDa LnK lane 4, is actually a small amount of spill over from lane 5.
60 kDa Tubulin
Mr (kDa) 1 2 3 4 5
97
FIGURE 15 Lnk Expression in Endothelial Cells Following Treatment with TNF a.
66
The Western blot was probed first with anti-Lnk antibody (AHP1003),
showing an increase in Lnk expression over time in lanes 3-5. It was then
45
stripped and reprobed with an anti-tubulin antibody to confirm loading
equivalence. Lane 1: Non-infected cells. Lane 2: Negative transfection
control-AdGFP. Lanes 3-5: 1, 2, and 3 hours post TNF treatment. 31

Densitometers and Analysis Software FIGURE 16 Detection of Rat Homer Protein Isoforms. Western blot of Homer isoforms
In order to make a permanent record, or to get an objective measure of the probed with anti-Homer [177-366] (AHP737) which detects the control and
signal generated on a Western blot, a densitometer is used to scan the blot isoform 1c. Lane 1: control, 30 mg rat cortical extract. Lane 2: untransfected
or film. Imaging software is then used to compare the signal generated control. Lane 3: Isoform 1a. Lane 4: Isoform 2b. Lane 5: Isoform 1c.
by the bands detected on the Western blot.

30 C hapte r 4 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 4 31

 Demonstrating Antibody Specificity prion disease. There are several ways to identify the presence of the abnormal
prion protein - a bioassay, immunohistochemistry on diseased tissue samples,
A simple way to demonstrate that the band seen on a Western blot is specific
and with a faster, well-characterized, and sensitive Western blot.
to the interaction between a target protein and a properly matched antibody
is by blocking the antibody from binding to the target epitope through The Western blot below shows Scrapie infected sheep brain lysates (Lanes
competition with a peptide that matches the sequence of the epitope. 3&4) compared to normal sheep brain lysates (Lanes 1&2). The key to this
AbD Serotec offers a range of blocking peptides for this purpose. particular Western assay is the use of Proteinase (PK) treatment on parallel
sets of samples. Since the Scrapie associated form of the prion protein is
The experiment is carried out during the antibody incubation stage
resistant to digestion, it is possible to distinguish between normal cellular
and compares antibody alone to antibody treated with the blocking
forms and abnormal prion protein in a sample based upon their sensitivity
peptide. If the peptide sequence correctly corresponds to the epitope
to PK. Thus, it is possible to discriminate diseased samples from normal ones
then the antibody will bind to the peptide and will not be available to bind
quickly and unambiguously on a Western blot. The absence of bands in lane 2
to the target protein on the blot. This technique would only be used with
indicates that the normal form is present as compared to the bands visible in
monoclonal antibodies, since they are directed against a single epitope,
lane 4, which indicate the presence of the pathogenic form.
and when the epitope is known. In the example below, lane 2 has no signal
because the peptide has effectively blocked the antibody from binding,
whereas in lane 1 the band is visible as expected.
Mr (kDa) 1 2 3 4
Mr (kDa) 1 2

102
81 30

46 25

33

30 12

FIGURE 17 Peptide Blocks Binding of Anti-CX3CR1 (AHP566) Antibody. Lane 1: THP-1

cell lysate (LYS008). Lane 2: THP-1 cell lysate + peptide to aa175-189 of
human CX3CR1. FIGURE 18 Detection of Scrapie Infected Sheep Brain. Bands in lane 4 indicate that
the protease resistant form of the prion protein is present in the sample
Alternate Forms of a Protein – Prion Disease as compared to normal tissue in lane 2. Lanes 1&2: Uninfected sheep brain
Prion disease is a rare, progressive, neurodegenerative disorder that homogenate. Lanes 3&4: Scrapie-infected sheep brain homogenate.
affects humans (vCJD, CJD), cows (BSE), sheep (Scrapie), and elk/deer (CWD). Lanes 1&3: No PK added. Lanes 2&4: Digested with PK.
It leads to characteristic brain lesions and a rapid loss in neurological
functions after a long latency period. The accumulation of an altered isoform
of a normal cellular protein appears to be instrumental in the development of

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 Buffers and Protocols
5

Chapter
Confirmation of HIV
In the diagnosis of HIV infection in patients, an ELISA is used first because
it demonstrates 99.5% specificity and is quick and easy to perform on
a large number of samples. Western blotting is used as a supplementary
assay because the ELISA is subject to false positives. In order to perform this
assay, patient serum samples are used as the source of antibodies for
immunodetection, and blots containing HIV antigens, proteins, viral lysates, Overview
or peptides are used as the source of target antigen on the Western blot.
The following buffers and methods provide a general starting point for use
If the HIV target proteins on the blot are detected by the patient serum then
with the majority of AbD Serotec reagents in Western blotting. When specific
it indicates that the patient sample is a true positive for HIV infection, since
recommendations are provided on product datasheets, those instructions
an immune response has been mounted by the patient. A similar strategy
should always be used instead of the general guidance offered here.
is also used in tests for Lyme disease and autoimmune disease.
With specialized laboratory equipment, such as for gel electrophoresis
and electroblotting, it is advisable to follow the manufacturer’s instructions.
If difficulties arise with any of our antibodies or related products, please feel
free to contact [email protected], as we may have additional
information on file and can assist with troubleshooting.

Buffers
N.B. Azide should not be added to any buffers that will be used with
HRP-labeled antibodies because it will inactivate the HRP enzyme.

PBS
8.0 g NaCl
0.2 g KCl
1.15 g Na2HPO4
0.2 g KH2PO4
Dissolve in 800 ml distilled water, adjust pH to 7.4, and then add more dH20
to a final volume of 1 liter. Sterilize by autoclaving and store at room
temperature (RT).

TBS
8.0 g NaCl
0.2 g KCl
3.0 g Tris base
Dissolve in 800 ml distilled water, adjust pH to 8.0 with 1 M HCl, and then
add more dH20 to a final volume of 1 liter. Sterilize by autoclaving and store at RT.

34 C hapte r 4 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 5 35

 10x TBS Stock: Electrophoresis and Transfer Buffers
500 mM Tris-HCl, pH 7.4 Laemmli 2x Sample Buffer
1.5 M NaCl 4% SDS
20% Glycerol
Cell Lysis Buffers
125 mM Tris, pH 6.8
NP-40 Lysis Buffer
0.02% Bromophenol blue
50 mM Tris, pH 8.0
200 mM DTT or 10% bME
150 mM NaCl
For best results DTT or bME is added fresh, just before use.
1% NP-40 (or Triton® X-100)
+ fresh protease inhibitors, see below Gel Electrophoresis Running Buffer

RIPA (Radio Immuno Precipitation Assay) Buffer 25 mM Tris base

50 mM Tris, pH 8.0 190 mM Glycine
150 mM NaCl 0.1% SDS
1% NP-40 (or Triton® X-100) Transfer Buffer
0.5% Sodium deoxycholate 50 mM Tris base
0.1% SDS 380 mM Glycine
+ fresh protease inhibitors, see below 0.1% SDS
Proteases Inhibitors 20% Methanol
Various proteases inhibitors are often added to lysis buffer to maintain the Ponceau S Stock Solution
integrity of the target protein when cells are disrupted. The following table
2% Ponceau S
provides some recommended working concentrations:
30% Trichloroacetic acid
Protease Inhibitors 30% Sulfosalicylic acid
Dilute 10-fold in distilled water prior to use.
Inhibitor Working Concentration Protease Inhibited

Aprotinin 1-2 mg/ml Trypsin, Chymotrypsin,

Washing Buffers
Plasmin, Kallikrein PBST
EDTA 1-2 mM Metalloproteases (Mg2+ PBS with 0.1% Tween® 20
and Mn2+)
TBST
EGTA 1 mM Metalloproteases (Ca2+)
TBS with 0.1% Tween® 20
Leupeptin 0.5-2 mg/ml Plasmin, Trypsin, Papain,
Cathepsin B Block Ace Wash Buffer (BUF029)
Pepstatin A 1 mg/ml Pepsin, Cathepsin D Reconstitute each 4 g vial in 100 ml distilled water. Dilute the reconstituted
PMSF (highly toxic) 50-100 mg/ml Serine and Cysteine
solution 10-fold, and add Tween® 20 to a final concentration of 0.05-0.2% v/v.
proteases

36 C hapte r 5 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 5 37

 Blocking Buffers Protocols
*Use BSA or Block Ace (BUF029) to block when probing with
anti-phosphoprotein antibodies, or for biotinylated primary Cell Lysis - Mammalian Cells
antibodies detected with an anti-biotin secondary. *For yeast and bacterial cell lysis consult Sambrook et al., or Harlow
and Lane.
5% Nonfat Dried Milk in PBST or TBST (Blotto/BLOTTO)
Add 5 g nonfat dried milk powder to 100 ml PBST or TBST. Adherent cells:
Dissolve with gentle stirring. Store at 4°C.
1.Wash cells directly in the tissue culture flask or dish by adding cold PBS
and rocking gently. Aspirate PBS and repeat. Keep tissue culture dish
3% BSA in PBST or TBST on ice throughout.
Dissolve 3 g of BSA Fraction V in 100 ml PBST or TBST with gentle stirring. 2. Add appropriate volume ice cold lysis buffer (with fresh protease
Store at 4°C. inhibitors), to the flask, approximately 1ml for a 100 mm tissue culture
dish. (Alternatively, cells can be removed from the flask with Trypsin-EDTA
Block Ace (BUF029) and then prepared using the suspension cell instructions below).
Reconstitute each 4 g vial in 100 ml distilled water, and use undiluted. 3. Incubate for 20 minutes on ice, and then scrape cells from the surface
using a rubber spatula.
4. Transfer to a microfuge tube and clarify the lysate by spinning
for 10 minutes at 12,000 RPM, at 4°C.
5. Transfer supernatant to a fresh tube and store on ice or frozen
at -20°C or -80°C.

Suspension cells:
1. Pipet cells into a fresh conical tube and place on ice.
2. Spin cells on low speed at 4°C, and aspirate off media.
3. Add 10 ml ice cold PBS, and gently invert tube to wash cells.
4. Spin cells on low speed, and aspirate off supernatant.
5. Repeat wash and aspiration. Resuspend cells in 5 ml ice cold PBS.
6. Count cells, and centrifuge on low speed at 4°C to form a cell pellet.
Aspirate off liquid.
7. Gently resuspend the cell pellet in ice cold cell lysis buffer (with fresh
protease inhibitors), use 1 ml buffer for 107 cells.
8. Incubate cells for 30 minutes on ice. If needed, sonicate the lysates
on ice for 15-30 seconds to disrupt genomic DNA and cellular components.
9. Transfer to a microfuge tube and clarify the lysate by spinning at 4°C,
for 10 minutes at 12,000 RPM.

38 C hapte r 5 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 5 39

 10. Decant the supernatant to a fresh tube, and discard cell pellet. Immunodetection – Indirect
Store on ice for immediate use, or at -20°C or -80°C until needed.
1. Place membrane into blocking solution for at least 2 hours at RT
11. Measure protein concentration with BCA, Lowry, or Bradford assays, or overnight at 4°C. Use a sufficient volume to keep the blot fully covered,
or by absorbance, prior to loading on a gel. with gentle agitation throughout.
2. Rinse the membrane briefly in washing buffer.
Tissue samples:
3. Incubate with primary antibody diluted in wash buffer or blocking buffer
1. Tissue samples are rinsed in cold PBS and placed in a tube (an antibody concentration of 1-10 µg/ml is generally acceptable).
on ice immediately following dissection. Incubate overnight at 4°C, or for 2 hours at room temperature.
2. For small samples (100 mg) chop into pieces with clean dissecting Use a sufficient volume to keep the blot fully covered, with gentle
instruments. Add tissue and 1-2 ml ice cold lysis buffer agitation throughout.
to dounce homogenizer, or sonicate in small tube. Homogenize
4. Rinse the membrane in wash buffer (3x10 minutes), with gentle agitation
or sonicate on ice.
and a sufficient volume to keep membrane well covered.
3. Clarify the lysate with a high speed spin in a microfuge at 4°C,
5. Add appropriate enzyme conjugated secondary antibody diluted in wash
for 10 minutes at 12,000 RPM.
buffer or blocking agent, and incubate for 1 hour at RT with gentle
4. Transfer supernatant to a fresh tube and discard cell pellet. agitation in a sufficient volume to ensure coverage.
Store on ice for immediate use, or at -20°C or -80°C until needed. (* No azide in the buffers if the secondary is labeled with HRP)
5. Measure protein concentration with BCA, Lowry, or Bradford assays, 6. Incubate the membrane with gentle agitation as follows: 4x5 minutes
or by absorbance, prior to loading on a gel. in wash buffer, followed by 2x5 minutes in PBS or TBS.

Gel Electrophoresis & Protein Transfer by Electroblotting 7. Drain excess PBS/TBS from the membrane and transfer to appropriate
enzyme substrate solution and incubate for time period recommended
* Detailed instructions for making gels in-house can be found in Sambrook et al. by manufacturer to visualize protein bands.
1. For standard denaturing and reducing conditions, mix sample 1:1 by volume
with Laemmli 2x sample buffer. Immunodetection – Direct
2. Heat to 100oC for 5 minutes or 70oC for 10 minutes. 1. Place membrane into blocking solution for at least 2 hours at RT,
3. Load 20-50 mg sample per lane, along with suitable positive or overnight at 4°C. Use a sufficient volume to keep the blot fully covered,
and negative controls. with gentle agitation throughout.
4. Run the gel and transfer to PVDF or nitrocellulose membrane according 2. Rinse the membrane briefly in washing buffer and then incubate
to the manufacturer’s instructions for the equipment and materials in use. with labeled primary antibody diluted in wash buffer or blocking buffer
5. Confirm protein transfer by staining for 1 minute with Ponceau S, followed (a concentration of 1-10 µg/ml is generally acceptable). Incubate overnight
by complete destain in distilled water. at 4°C, or for 2 hours at room temperature. Use a sufficient volume to keep
6. Mark position of molecular weight standards if they are unlabeled. the blot fully covered, with gentle agitation throughout.
3. Wash the membrane in a sufficient volume and with gentle agitation
(For our in-house Western blotting, we use NuPAGE® Novex® precast 4-12%
as follows: 4x5 minutes in wash buffer and 2x5 minutes in PBS or TBS.
gradient gels run in Tris-Acetate buffer and transferred to nitrocellulose using
the Xcell SureLockTM II blot module from Invitrogen, as per the manufacturer’s 4. Drain excess PBS/TBS from the membrane and transfer to appropriate
instructions.) enzyme substrate solution and incubate for time period recommended
by manufacturer to visualize protein bands.

40 C hapte r 5 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 5 41

 Troubleshooting
6
Unusual or Unexpected Bands

Chapter
Difference seen Possible Cause Action/Solution

Band(s) at lower T arget protein has been cleaved Use a fresh sample which has been
molecular weight than or digested kept on ice
expected Splice variants exist Add fresh protease inhibitors
Another protein bearing the to the lysis buffer
Overview same/similar epitope has been Try alternate antibody
detected by antibody
While Western blotting is a relatively simple and straightforward technique,
Band(s) at slightly Protein may be glycosylated Use enzymes to remove suspected
it does not always yield results that meet with expectations. When this higher molecular or otherwise modified at one modification returning molecular
occurs, it is useful to be able to quickly isolate the possible causes and weight than expected, or more amino acid residues weight closer to expected
to formulate an effective solution by troubleshooting the experiment. In this and may be blurred Check amino acid sequence
chapter, suggestions are offered to assist in identifying and resolving some and literature
frequently encountered problems that arise during the course of Western Band(s) at significantly Dimers, multimers, or protein- Add fresh DTT or bME to samples and
higher molecular protein interactions may be reheat before repeating experiment
blotting. Many of these problems can be avoided with careful attention
weight than expected occurring because samples Prepare new samples with fresh
to experimental protocols, and with optimization at key stages throughout have not been fully reduced and loading buffer
the procedure. As mentioned at the end of Chapter 3, the proper antibody denatured
concentration can be determined with the use of dot blots and slot blots, Multiple bands Primary antibody concentration Use an affinity-purified primary
significantly reducing the need for future troubleshooting. Additionally, at various molecular may be too high, or there antibody
the use of negative and positive controls are of great help in assessing where weights is a cross-reactivity with similar Optimize primary antibody
epitopes on other proteins concentration
things have gone wrong, and eliminating some possible sources of error Try another antibody
under consideration. Check antibody specificity with
blocking peptide
Since one issue that commonly arises during Western blotting is the presence Secondary antibody Decrease/optimize the concentration
of unusual or unexpected bands on the blot, our troubleshooting section concentration is too high leading of the secondary antibody
begins with a table describing some common reasons and potential solutions to non-specific binding Use an affinity-purified secondary
for addressing and evaluating this type of problem. antibody
Repeat immunodetection with
In most cases, the rest of the troubleshooting issues can be grouped into secondary antibody alone to check
for non-specific binding
three major types: no bands, faint bands, and signal on Western blots that
Protein exists in several different Check literature
interferes with bands. Therefore, the potential causes and solutions have been
isoforms
organized in this manner, and also with regard to potential problem sources, Bands are blurry Gel was run at too high a voltage Repeat gel at lower voltage
i.e. antibody, antigen, technique, or buffer related where applicable. For Incorrect running buffer Prepare fresh running buffer
further assistance when using our products in Western blotting, contact composition
[email protected]. Trapped air bubble present Carefully remove air bubbles between
during transfer the gel and the membrane before
protein transfer
Bands are smile Running conditions were too fast Check and optimize gel
shaped, not flat so gel became over heated electrophoresis conditions
Run gel at 4°C
White (negative) bands Too much protein has been Load less sample
on the film when using loaded Repeat with dilution series of sample
ECL detection Antibody concentration Reduce/optimize the antibody
is too high concentrations

42 C hapte r 6 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 6 43

 No Bands Faint Bands or Weak Signal
Source of Problem Possible Cause Action/Solution Source of Problem Possible Cause Action/Solution

Antibody related Inappropriate secondary Retrace steps to check compatibility Antibody related Primary or secondary antibody Repeat using higher concentration
antibody used between primary and secondary antibodies concentrations were too low of antibody
Reprobe with correct secondary or strip blot Optimize antibody concentration with
and reprobe if necessary dot blots
Repeat experiment with the correct
antibody combination Low antigen-antibody Reduce the number of wash steps
Wrong concentration of Increase the antibody concentration binding affinity to a minimum
antibody or low affinity to the 2-4 fold higher than initially recommended Increase the antibody concentration
target protein Increase length of incubation 2-4 fold higher than the recommended
Test/optimize antibody on dot blots starting dilution
Try another antibody
Antigen related Insufficient sample loaded on Check concentration of sample
Antibody not suitable C
 heck datasheet for recommended conditions the gel Increase the amount of source material
for Western blotting Test/optimize antibody on dot blots Immunoprecipitate, fractionate,
Try alternate antibody or concentrate sample
Antibody has lost activity Test on a dot blot at several concentrations
due to long term or improper Use fresh aliquot of antibody that has been Blot has been stripped Redo blot since antigen may have been
storage stored at -20°C or below and reprobed stripped off or damaged by stripping
Antigen related Antigen not expressed Use another source of target protein process
in the source material Technique related Low transfer efficiency Confirm transfer with Ponceau S staining
Not enough antigen loaded Check concentration of sample Optimize transfer conditions for target
on the gel Increase the amount of source material protein size
Immunoprecipitate, fractionate, Optimize transfer buffers for methanol
or concentrate the sample and SDS concentrations
Technique related Transfer did not work properly  onfirm protein transfer by staining the
C Film exposure time (ECL Test different exposure times
membrane with Ponceau S and/or the gel detection) was too short
with Coomassie dye.
N
 ote how well any prestained molecular Buffer related Non-fat dry milk may mask Decrease % of milk in the blocking
weight markers have transferred onto the blot some antigens and antibody solutions
Optimize/check transfer conditions and set Try alternate blocking solution
up (especially orientation to electrodes)
Repeat using two membranes in case
protein has transferred through the first
Washes are too stringent Use fewer washers
Reduce washing time
Buffer related Blocking agent is interfering Try lower concentration
with signal Try alternate blocking agent
Buffers may contain sodium Use azide free buffers
azide which inactivates HRP
Peroxide may be inactive Add fresh peroxide to substrate buffer
reducing activity of peroxidase
ECL detection reagents have Use fresh detection reagents
been contaminated

44 C hapte r 6 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 6 45

 High Background Signal on the Blot Patchy or Uneven Spots on the Blot
Source of Problem Possible Cause Action/Solution Source of Problem Possible Cause Action/Solution

Antibody related Concentration of primary and/or Reduce/optimize the antibody Antibody related HRP-linked secondary antibody Spin secondary antibody and filter
secondary antibody too high concentrations has aggregated to remove aggregates
Reduce/optimize incubation times
Use affinity purified antibodies Technique related Not enough solution used during Make sure the membrane is fully
Primary and/or secondary binds Use alternate blocking solution incubation and/or washing immersed and agitated throughout
to the blocking agent incubations
Antibody has reduced activity due U
 se a fresh aliquot of antibody that Trapped air bubbles Carefully remove air bubbles between
to long term or improper storage has been stored at -20°C or below the gel and the membrane before
Phospho-specific antibody U
 se alternate blocking solution- BSA protein transfer
has reacted with casein or Block Ace (AbD Serotec BUF029)
(a phosphoprotein) in nonfat Uneven agitation during Use a shaker for all incubations
milk blocking agent incubation
Secondary antibody is binding Try another blocking agent Buffer related Antibodies are binding to the Filter the blocking agent
to blocking agent Reduce protein concentration blocking agent Try another blocking agent
of blocking agent
Test secondary antibody alone on blot
Try alternate form of secondary,
such as F(ab’)2
Antigen related Non-specific interaction with Sonicate the lysates
genomic DNA in samples Add DNAse to lysis buffer
Technique related Membrane blocking is C
 onfirm concentration matches
not sufficiant that recommended in the protocol
Try other blocking agents
Try alternate incubation temperatures,
including room temperature
Membrane has dried out Repeat procedure taking care that the
blot does not dry out during any step
by using sufficient volumes and
agitation throughout
PVDF membrane has higher Try nitrocellulose instead
background
Inadequate washing between Increase the length of washing steps
incubations Use a larger volume of wash buffer
Incubation temperature too high Try alternate incubation temperatures
such as 4°C
Film over-exposed or blot Wait 5-10 minutes and then re-expose
developed for too long blot to film
Reduce exposure and/or development
times
Buffer related Insufficient concentration Use TBS containing >0.1% Tween® 20
of detergent in the buffers Try stronger detergent, such as NP-40
Transfer, incubation, or blocking Use fresh buffers
solutions are contaminated

46 C hapte r 6 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om Chapter 6 47

 References and Further Reading Acknowledgements
B olt, M. and Mahoney, P. (1997) High-efficiency blotting of proteins of diverse sizes The author gratefully recognizes Joanne Martin, PhD., Clare Desforges,
following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. MSc., and Susan P. Fairchild, PhD. for their key contributions and valuable
247, 185-192 assistance.
Burnette, W.N. (1981) “Western Blotting”: Electrophoretic transfer of proteins from sodium
dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic Thanks to the following for images and data:
detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195-203 Figure 2 & 6: Joanne Martin, PhD., AbD Serotec, Oxford, UK
Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York
Figure 7 & 9: Joanna Rowe, PhD., and John Woodley, AbD Serotec,
Oxford, UK
Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York Figure 12: Dra. Ainoha García Claver, Unidad de Patología Molecular,
Towbin, H., et al., (1979) Electrophoretic transfer of proteins from polyacrylamide gels Hospital Virgen de la Salud, Toledo, Spain, and Dra. Barbara Meléndez,
to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. Hospital Virgen de la Salud, Toledo, Spain
76, 4350-4354
Figure 15: Juliette Fitau, Institut National de la Santé et de la Recherche
Towbin, H. and Gordon, J. (1984) Immunoblotting and dot immunobinding--current status Médicale (INSERM), and Institut de Transplantation et de Recherche en
and outlook. J. Immunol. Methods 72, 313-340 Transplantation (ITERT), Nantes, France
Wadsworth, J. D. et al., (2001) Tissue distribution of protease resistant prion protein in
variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet Figure 16: Will Charles and Adam Podmore, Auckland UniServices Ltd.,
358, 171-180 Auckland, New Zealand

Figure 18: Dr Sandra McCutcheon and Boon Chin Tan, Neuropathogenesis

Division, The Roslin Institute, UK

HuCAL® and LYNX Rapid Conjugation Kit® are registered trademarks of MorphoSys UK Ltd.
HuCAL® products are for in vitro use only. All AbD Serotec reagents are for research purposes
only, not for therapeutic or diagnostic use.

DyLight® dyes are a registered trademark of Thermo Fisher Scientific and its subsidiaries. LI-COR®
and Odyssey® Blocking Buffer are registered trademarks of LI-COR Biosciences. NuPAGE®
and Novex® are registered trademarks of Invitrogen.

48 Your fi rs t choi ce for anti bodi es ! www.abds erot e c . c om

 Your first choice for antibodies!
Over 14,000 performance-guaranteed reagents
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