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A ring of lymphoid tissue in humans that is composed
of the lingual tonsil, palatine tonsils and the nasopharyngeal tonsils.
Mucosa-Associated Lymphoid Tissue
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Warm Autoantibodies
Autoantibodies that react as well, or more strongly, at
37 °C than lower temperatures.
Hemolytic Anemia, Autoimmune
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Western Blot Analysis
Georg Brunner
Fachklinik Hornheide an der Universität Münster
Dorbaumstrasse 300
D-48157 Münster
Germany
Synonyms
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Immunoblotting,
electroblotting, protein blotting.
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Short Description
Western blotting is a protein analysis technique which
combines protein separation in an electric field with
immunochemical methods of protein detection. It is
used to identify, characterize, quantify, and/or isolate
proteins or antibodies present in complex biological
samples. First, proteins are separated according to
their molecular size and/or net charge by electrophoretic migration through three-dimensional polyacrylamide gels. This is followed by the electrophoretic
transfer of the proteins out of the gel onto a two-dimensional, protein-binding membrane support. Finally, proteins bound to the membrane are visualized by
the binding of labeled, protein-specific antibodies or
ligands.
Characteristics
Analogous to the membrane blotting techniques for
DNA (Southern blotting) or RNA (Northern blotting),
electrophoretic blotting and analysis of proteins on a
membrane support was developed and termed Western
blotting (1,2). Western blot analysis allows the identification of proteins in complex mixtures as well as
their characterization, i.e. determination of quantity,
molecular size, net charge, epitopes for antibody recognition, or ligand binding sites. It is also a particularly versatile technique to identify the presence, quantity, and specificity of antibodies, e.g. polyclonal antibodies present in serum samples.
The sensitivity of protein detection is in the range of
10–100 fmoles (1–10 ng of protein). The sensitivity is
mainly determined by the specificity (signal-to-noise
ratio) and sensitivity of the detection system, but also
depends on the protein loading capacity of electrophoresis, the efficiency of protein transfer, and the concentration of the proteins of interest in the sample.
Western blotting involves a number of different steps
(Figure 1), such as the separation of the proteins by
electrophoretic techniques (I), the electrophoretic
transfer of the separated proteins onto a membrane
support (II), the blocking of free protein binding
sites on the membrane (III), and the detection of the
proteins of interest using specific antibodies or ligands
(IV).
(I) Proteins in complex biological samples such as cell
or tissue extracts are diluted into sodium dodecylsulfate(SDS)-containing gel electrophoresis sample buffer. Alternatively, cell or tissue samples can be extracted directly in sample buffer followed by solubilization of the proteins by boiling.
Electrophoresis is usually performed using discontinuous standard Tris/glycine SDS-polyacrylamide
( SDS-PAGE) gels (3), separating the proteins present in the sample based on their relative molecular
size. However, several other techniques are possible
and have been used to increase the resolution and
versatility of electrophoretic protein separation. Thus,
polyacrylamide gradient gels enhance the resolution of
proteins in crude cell or tissue lysates over a much
wider size range than standard gels of uniform concentration. Proteomics analysis requires maximum
protein resolution, and complex mixtures of proteins
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Waldeyer’s Ring
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Western Blot Analysis
Western Blot Analysis. Figure 1 Western blot analysis.
are therefore subjected to 2-D gel electrophoresis
which can easily resolve 1500 different proteins. Proteins are separated, in the first dimension, by isoelectric focusing according to their isoelectric point, followed by separation, in the second dimension, by
SDS-PAGE on the basis of their molecular size.
(II) Following electrophoretic separation, proteins are
transferred out of the 3-D polyacrylamide gel matrix
onto a 2-D membrane support in order to render them
accessible for antibody recognition or ligand binding
(3). Whereas DNA and RNA are efficiently blotted by
capillary diffusion, electrophoretic elution is the preferred technique for protein blotting. The membrane
is placed in direct contact with the polyacrylamide gel
in an electric field, which induces the migration of the
SDS-loaded, negatively charged proteins out of the gel
and onto the membrane. Alternatively, particularly
when ultra-thin gels are being used, proteins can be
transferred simply by diffusion (diffusion blotting).
Protein transfer by diffusion is less efficient as compared to electrophoretic transfer, but efficiency can be
significantly increased by raising the temperature to
70°C.
Typical blotting membrane materials are nitrocellulose, polyvinylidene difluoride (PVDF), and nylon,
which vary in their physical properties and protein
binding characteristics and capacity.
Electrophoretic protein transfer can be performed by
immersing the gel-membrane sandwich in a blotting
chamber in an appropriate buffer to produce the desired electric field strength (wet transfer). Alternatively, protein elution can be achieved by placing the
sandwich, between filter paper soaked with the respective transfer buffer, in direct contact with two plate
electrodes (semi-dry transfer). While the latter procedure is considerably faster than the wet transfer, it is
not very efficient for larger proteins (> 100 kDa) due
to the limited time of transfer and electric field
strength that can be applied.
The efficiency of the protein transfer depends on the
pore size of the polyacrylamide gel and the efficiency
of the protein binding to the blotting membrane. While
small proteins (< 20 kDa) easily migrate out of the gel,
their binding to the membrane is often inefficient and
requires a membrane with a small pore size as well as
methanol in the transfer buffer. Methanol, however,
shrinks the gel, thus decreasing the pore size of the
gel and further impairing protein transfer. On the other
hand, large linear molecules, such as proteoglycans,
are often difficult to transfer and require high electrophoretic field strength and cooling as well as extended
transfer time periods (24–48 hours). Addition of SDS
to the buffer enhances the transfer of proteins out of
the gel but can interfere with protein binding to the
membrane support.
Following transfer, the protein pattern blotted onto the
membrane can be visualized, on a duplicate blot run in
parallel, using protein stains such as india ink (irreversible) or Ponceau S (reversible).
(III) Following protein transfer, free protein-binding
sites on the membrane support are blocked by incubation with a concentrated protein solution such as bovine serum albumin, non-fat dried milk, or casein, or
with a detergent such as Tween 20. This prevents non-
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Western Blot Analysis
specific interactions of the detection system with the
membrane support during subsequent incubation
steps.
(IV) Proteins of interest are most commonly detected
via the recognition of antigenic epitopes by specific
antibodies. This requires the epitopes to be resistant to
denaturation. Polyclonal antisera are more likely to
contain antibodies reacting with denatured antigens
than monoclonal antibodies. To control for non-specific antibody-protein interactions, pre-immune or irrelevant antibodies of the same species are applied on a
duplicate blot run in parallel. Alternatively, protein
receptors blotted onto the membrane can be detected
via the binding of specific ligands.
Binding to defined protein bands is visualized either
by using directly labeled primary antibodies or ligands, or by incubating the membrane with unlabeled
primary followed by labeled secondary antibodies.
Antibodies are either radioactively labeled with 125iodine (detection by radiography) or conjugated to enzymes such as alkaline phosphatase or horseradish
peroxidase (detection by enzyme assay using chromogenic or chemoluminogenic substrates). The sensitivity of detection can be enhanced by labeling the secondary antibodies with biotin, followed by incubation
with labeled streptavidin, thus amplifying the detection system.
Western blotting analysis can be combined with other
techniques to enhance the sensitivity of protein detection or to purify antibodies. To enhance the sensitivity
(i.e. the signal-to-noise ratio) the proteins of interest
can be concentrated and enriched prior to electrophoresis. This is most commonly done by immunoprecipitation using specific antibodies bound to protein Aagarose beads. To affinity-purify antigen-specific antibodies, the respective protein bands can be excised,
following incubation with a polyclonal antiserum, and
bound antibodies are eluted from the membrane pieces
at an acidic pH.
Pros and Cons
Western blot analysis combines the high resolution
power of protein electrophoresis with the sensitivity
and specificity of antibody-antigen or receptor-ligand
interactions.
Sample preparation does not require protein purification, since crude biological samples can be subjected
directly to SDS-PAGE. In addition, problems of protein insolubility or instability are overcome by extracting or diluting cells or tissue samples in SDS-containing electrophoresis sample buffer, followed by a boiling step which inactivates protease activities potentially present in the sample.
Samples can be selectively labeled for western blot
analysis in order to allow for the specific analysis of
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defined protein fractions, for example by biotinylation
of cell surface proteins prior to extraction.
Protein detection following electrophoretic transfer
can be modified in order to visualize proteins bound
to the membrane based on their functional activity,
instead of via the recognition by specific antibodies.
For example, blotted receptors can be detected by the
specific binding of labeled ligands.
Proteins are usually denatured during sample preparation, electrophoresis, or electrophoretic transfer, which
can interfere with their detection on the membrane and
does not allow further functional characterization of
the blotted proteins.
Although the signal obtained in western blot analysis
correlates with the protein amount present on the
membrane, at least over a certain range of protein concentration, detection is only semi-quantitative. Protein
transfer efficiency out of the SDS-PAGE gel as well as
protein binding to the membrane support are influenced by the size, the conformation, and other physical properties of the proteins and the membrane. In
addition, the linear relationship between protein
amount and signal obtained depends on the linearity
of the detection system and is limited by the protein
binding capacity of the membrane.
Detection of minor proteins in complex biological
samples can be difficult due to non-specific hydrophobic or electrostatic protein-protein interactions of antibodies or ligands with other proteins present in the
sample. In this case, partial purification of the proteins
is required, for example by immunoprecipitation, protein chromatography, of cell/tissue fractionation prior
to electrophoresis.
Predictivity
Western blotting allows the fast and sensitive qualitative and semi-quantitative analysis of complex protein
mixtures as well as the identification of antigen-specific antibodies in serum. However, the results
strongly depend on the specificity and sensitivity of
the detection system as well as on the antigenic and
physical properties of the proteins under investigation.
Therefore, the preservation of antigenic epitopes or
ligand binding sites, as well as the efficiency of protein
transfer, need to be verified, and the detection system
requires standardization to allow quantification of the
results. Proteolytic cleavage of proteins in the sample
prior to electrophoresis, resulting in protein fragments
still containing the epitope for antibody recognition,
can complicate the interpretation of the results.
Relevance to Humans
Western blot analysis is widely used as a tool for the
analysis of specific proteins as well as for broader
proteomic approaches (particularly in combination
with 2-D electrophoretic techniques), allowing the
W
White Pulp
qualitative and quantitative analysis of complex biological samples, e.g. crude cell and tissue extracts or
plasma or serum samples. It can be used for protein
screening, quantification, as well as for antibody isolation.
Western blotting has important clinical applications,
like the confirmatory test for human immunodeficiency virus type 1 (HIV-1) infection. Viral proteins are
separated electrophoretically, transferred onto a membrane support, and incubated with patient sera. Binding of serum antibodies to defined viral antigens is
then visualized using Western blot analysis.
Regulatory Environment
lulose sheets: Procedure and some applications. Proc Natl
Acad Sci USA 76:4350–4354
3. Laemmli UK (1970) Cleavage of the structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227:680–685
White Pulp
Part of the spleen around central arterioles where lymphoid cells reside. Comprises three major compartments: the periarteriolar lymphocyte sheath or PALS,
follicles and marinal zone.
Spleen
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Not applicable.
1. Burnette WN (1981) Western blotting: Electrophoretic
transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated
protein A. Anal Biochem 112:195–203
2. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocel-
WHO
World Health Organization. The WHO has responsibility for regulating vaccines.
Immunotoxicology
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References