The western blot is a widely used analytical technique used to detect specific proteins
in a sample of tissue homogenate or extract.
It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins
by the length of the polypeptide.
The proteins are then transferred to a membrane, where they are stained with antibodies specific
to the target protein.
The gel electrophoresis step is included in Western blot analysis to resolve the issue
of the cross-reactivity of antibodies.
An improved immunoblot method, Zestern analysis, is able to address this issue without the
electrophoresis step, thus significantly improving the efficiency of protein analysis.
There are now many reagent companies that specialize in providing antibodies against
tens of thousands of different proteins.
Commercial antibodies can be expensive, although the unbound antibody can be reused between
experiments.
This method is used in the fields of molecular biology, biochemistry, immunogenetics and
other molecular biology disciplines.
Other related techniques include dot blot analysis, zestern analysis, immunohistochemistry
where antibodies are used to detect proteins in tissues and cells by immunostaining, and
enzyme-linked immunosorbent assay.
The method originated in the laboratory of Harry Towbin at the Friedrich Miescher Institute.
The name western blot was given to the technique by W. Neal Burnette and is a play on the name
Southern blot, a technique for DNA detection developed earlier by Edwin Southern.
Detection of RNA is termed northern blot and was developed by George Stark at Stanford.
Steps Tissue preparation
Samples can be taken from whole tissue or from cell culture.
Solid tissues are first broken down mechanically using a blender, using a homogenizer, or by
sonication.
Cells may also be broken open by one of the above mechanical methods.
However, virus or environmental samples can be the source of protein and thus western
blotting is not restricted to cellular studies only.
Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and
to solubilize proteins.
Protease and phosphatase inhibitors are often added to prevent the digestion of the sample
by its own enzymes.
Tissue preparation is often done at cold temperatures to avoid protein denaturing and degradation.
A combination of biochemical and mechanical techniques – comprising various types of
filtration and centrifugation – can be used to separate different cell compartments and
organelles.
Gel electrophoresis
The proteins of the sample are separated using gel electrophoresis.
Separation of proteins may be by isoelectric point, molecular weight, electric charge,
or a combination of these factors.
The nature of the separation depends on the treatment of the sample and the nature of
the gel.
This is a very useful way to identify a protein.
By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded
with sodium dodecyl sulfate.
SDS-PAGE maintains polypeptides in a denatured state once they have been treated with strong
reducing agents to remove secondary and tertiary structure and thus allows separation of proteins
by their molecular weight.
Sampled proteins become covered in the negatively charged SDS and move to the positively charged
electrode through the acrylamide mesh of the gel.
Smaller proteins migrate faster through this mesh and the proteins are thus separated according
to size.
The concentration of acrylamide determines the resolution of the gel - the greater the
acrylamide concentration the better the resolution of lower molecular weight proteins.
The lower the acrylamide concentration the better the resolution of higher molecular
weight proteins.
Proteins travel only in one dimension along the gel for most blots.
Samples are loaded into wells in the gel.
One lane is usually reserved for a marker or ladder, a commercially available mixture
of proteins having defined molecular weights, typically stained so as to form visible, coloured
bands.
When voltage is applied along the gel, proteins migrate through it at different speeds dependent
on their size.
These different rates of advancement separate into bands within each lane.
It is also possible to use a two-dimensional gel which spreads the proteins from a single
sample out in two dimensions.
Proteins are separated according to isoelectric point in the first dimension, and according
to their molecular weight in the second dimension.
Transfer In order to make the proteins accessible to
antibody detection they are moved from within the gel onto a membrane made of nitrocellulose
or polyvinylidene difluoride.
The primary method for transferring the proteins is called electroblotting and uses an electric
current to pull proteins from the gel into the PVDF or nitrocellulose membrane.
The proteins move from within the gel onto the membrane while maintaining the organization
they had within the gel.
An older method of transfer involves placing a membrane on top of the gel, and a stack
of filter papers on top of that.
The entire stack is placed in a buffer solution which moves up the paper by capillary action,
bringing the proteins with it.
In practice this method is not used as it takes too much time; electroblotting is preferred.
As a result of either "blotting" process, the proteins are exposed on a thin surface
layer for detection.
Both varieties of membrane are chosen for their non-specific protein binding properties.
Protein binding is based upon hydrophobic interactions, as well as charged interactions
between the membrane and protein.
Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not
stand up well to repeated probings.
The uniformity and overall effectiveness of transfer of protein from the gel to the membrane
can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes.
Ponceau S is the more common of the two, due to its higher sensitivity and water solubility,
the latter making it easier to subsequently destain and probe the membrane, as described
below.
Blocking Since the membrane has been chosen for its
ability to bind protein and as both antibodies and the target are proteins, steps must be
taken to prevent the interactions between the membrane and the antibody used for detection
of the target protein.
Blocking of non-specific binding is achieved by placing the membrane in a dilute solution
of protein - typically 3-5% Bovine serum albumin or non-fat dry milk in Tris-Buffered Saline
or I-Block, with a minute percentage of detergent such as Tween 20 or Triton X-100.
The protein in the dilute solution attaches to the membrane in all places where the target
proteins have not attached.
Thus, when the antibody is added, there is no room on the membrane for it to attach other
than on the binding sites of the specific target protein.
This reduces "noise" in the final product of the western blot, leading to clearer results,
and eliminates false positives.
Detection During the detection process the membrane
is "probed" for the protein of interest with a modified antibody which is linked to a reporter
enzyme; when exposed to an appropriate substrate this enzyme drives a colourimetric reaction
and produces a color.
For a variety of reasons, this traditionally takes place in a two-step process, although
there are now one-step detection methods available for certain applications.
Two steps Primary antibody
The primary antibodies are generated when a host species or immune cell culture is exposed
to protein of interest.
Normally, this is part of the immune response, whereas here they are harvested and used as
sensitive and specific detection tools that bind the protein directly.
After blocking, a dilute solution of primary antibody is incubated with the membrane under
gentle agitation.
Typically, the solution is composed of buffered saline solution with a small percentage of
detergent, and sometimes with powdered milk or BSA.
The antibody solution and the membrane can be sealed and incubated together for anywhere
from 30 minutes to overnight.
It can also be incubated at different temperatures, with higher temperatures being associated
with more binding, both specific and non-specific.
Secondary antibody After rinsing the membrane to remove unbound
primary antibody, the membrane is exposed to another antibody, directed at a species-specific
portion of the primary antibody.
Antibodies come from animal sources; an anti-mouse secondary will bind to almost any mouse-sourced
primary antibody, which allows some cost savings by allowing an entire lab to share a single
source of mass-produced antibody, and provides far more consistent results.
This is known as a secondary antibody, and due to its targeting properties, tends to
be referred to as "anti-mouse," "anti-goat," etc.
The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline
phosphatase or horseradish peroxidase.
This means that several secondary antibodies will bind to one primary antibody and enhance
the signal.
Most commonly, a horseradish peroxidase-linked secondary is used to cleave a chemiluminescent
agent, and the reaction product produces luminescence in proportion to the amount of protein.
A sensitive sheet of photographic film is placed against the membrane, and exposure
to the light from the reaction creates an image of the antibodies bound to the blot.
A cheaper but less sensitive approach utilizes a 4-chloronaphthol stain with 1% hydrogen
peroxide; reaction of peroxide radicals with 4-chloronaphthol produces a dark purple stain
that can be photographed without using specialized photographic film.
As with the ELISPOT and ELISA procedures, the enzyme can be provided with a substrate
molecule that will be converted by the enzyme to a colored reaction product that will be
visible on the membrane.
Another method of secondary antibody detection utilizes a near-infrared fluorophore-linked
antibody.
Light produced from the excitation of a fluorescent dye is static, making fluorescent detection
a more precise and accurate measure of the difference in signal produced by labeled antibodies
bound to proteins on a western blot.
Proteins can be accurately quantified because the signal generated by the different amounts
of proteins on the membranes is measured in a static state, as compared to chemiluminescence,
in which light is measured in a dynamic state.
A third alternative is to use a radioactive label rather than an enzyme coupled to the
secondary antibody, such as labeling an antibody-binding protein like Staphylococcus Protein A or Streptavidin
with a radioactive isotope of iodine.
Since other methods are safer, quicker, and cheaper, this method is now rarely used; however,
an advantage of this approach is the sensitivity of auto-radiography based imaging, which enables
highly accurate protein quantification when combined with optical software.
One step Historically, the probing process was performed
in two steps because of the relative ease of producing primary and secondary antibodies
in separate processes.
This gives researchers and corporations huge advantages in terms of flexibility, and adds
an amplification step to the detection process.
Given the advent of high-throughput protein analysis and lower limits of detection, however,
there has been interest in developing one-step probing systems that would allow the process
to occur faster and with fewer consumables.
This requires a probe antibody which both recognizes the protein of interest and contains
a detectable label, probes which are often available for known protein tags.
The primary probe is incubated with the membrane in a manner similar to that for the primary
antibody in a two-step process, and then is ready for direct detection after a series
of wash steps.
Analysis After the unbound probes are washed away,
the western blot is ready for detection of the probes that are labeled and bound to the
protein of interest.
In practical terms, not all westerns reveal protein only at one band in a membrane.
Size approximations are taken by comparing the stained bands to that of the marker or
ladder loaded during electrophoresis.
The process is commonly repeated for a structural protein, such as actin or tubulin, that should
not change between samples.
The amount of target protein is normalized to the structural protein to control between
groups.
A superior strategy is the normalization to the total protein visualized with trichloroethanol
or epicocconone.
This practice ensures correction for the amount of total protein on the membrane in case of
errors or incomplete transfers.
Colorimetric detection The colorimetric detection method depends
on incubation of the western blot with a substrate that reacts with the reporter enzyme that
is bound to the secondary antibody.
This converts the soluble dye into an insoluble form of a different color that precipitates
next to the enzyme and thereby stains the membrane.
Development of the blot is then stopped by washing away the soluble dye.
Protein levels are evaluated through densitometry or spectrophotometry.
Chemiluminescent detection Chemiluminescent detection methods depend
on incubation of the western blot with a substrate that will luminesce when exposed to the reporter
on the secondary antibody.
The light is then detected by CCD cameras which capture a digital image of the western
blot or photographic film.
The use of film for western blot detection is slowly disappearing because of non linearity
of the image.
The image is analysed by densitometry, which evaluates the relative amount of protein staining
and quantifies the results in terms of optical density.
Newer software allows further data analysis such as molecular weight analysis if appropriate
standards are used.
The main companies offering Chemiluminescence imaging systems are Vilber Lourmat, GE and
Biorad.
Radioactive detection Radioactive labels do not require enzyme substrates,
but rather allow the placement of medical X-ray film directly against the western blot
which develops as it is exposed to the label and creates dark regions which correspond
to the protein bands of interest.
The importance of radioactive detections methods is declining due to its hazardous radiation,
because it is very expensive, health and safety risks are high, and ECL provides a useful
alternative.
Fluorescent detection The fluorescently labeled probe is excited
by light and the emission of the excitation is then detected by a photosensor such as
CCD camera equipped with appropriate emission filters which captures a digital image of
the western blot and allows further data analysis such as molecular weight analysis and a quantitative
western blot analysis.
Fluorescence is considered to be one of the best methods for quantification, but is less
sensitive than chemiluminescence.
Secondary probing One major difference between nitrocellulose
and PVDF membranes relates to the ability of each to support "stripping" antibodies
off and reusing the membrane for subsequent antibody probes.
While there are well-established protocols available for stripping nitrocellulose membranes,
the sturdier PVDF allows for easier stripping, and for more reuse before background noise
limits experiments.
Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol
or methanol before use.
PVDF membranes also tend to be thicker and more resistant to damage during use.
2-D gel electrophoresis
2-dimensional SDS-PAGE uses the principles and techniques outlined above.
2-D SDS-PAGE, as the name suggests, involves the migration of polypeptides in 2 dimensions.
For example, in the first dimension polypeptides are separated according to isoelectric point,
while in the second dimension polypeptides are separated according to their molecular
weight.
The isoelectric point of a given protein is determined by the relative number of positively
and negatively charged amino acids, with negatively charged amino acids contributing to a low
isoelectric point and positively charged amino acids contributing to a high isoelectric point.
Samples could also be separated first under nonreducing conditions using SDS-PAGE and
under reducing conditions in the second dimension, which breaks apart disulfide bonds that hold
subunits together.
SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel.
In principle, this method allows for the separation of all cellular proteins on a single large
gel.
A major advantage of this method is that it often distinguishes between different isoforms
of a particular protein - e.g. a protein that has been phosphorylated.
Proteins that have been separated can be cut out of the gel and then analysed by mass spectrometry,
which identifies the protein.
Please refer to reference articles for examples of the application of 2-D SDS PAGE.
Medical diagnostic applications The confirmatory HIV test employs a western
blot to detect anti-HIV antibody in a human serum sample.
Proteins from known HIV-infected cells are separated and blotted on a membrane as above.
Then, the serum to be tested is applied in the primary antibody incubation step; free
antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added.
The stained bands then indicate the proteins to which the patient's serum contains antibody.
A western blot is also used as the definitive test for Bovine spongiform encephalopathy.
Some forms of Lyme disease testing employ western blotting.
Western blot can also be used as a confirmatory test for Hepatitis B infection.
In veterinary medicine, western blot is sometimes used to confirm FIV+ status in cats.
Protocols Western Blot Video Protocol
Western Blotting Protocol from Protocolmonkey Modified western blotting protocols from Biotechniques
Dr. Mark Barton Frank Lab protocol Kamps's Western Blotting Protocol
[1] CSH Protocols collection: Immunoblotting
Western Blot Protocol from HowtoWesternBlot.net See also
Far-Eastern blotting Far-western blotting
Eastern blot Northwestern blot
SDS-PAGE Fast parallel proteolysis [2]
Zestern analysis References
External links Western blot analysis of sub-cellular fractionated
samples using the Odyssey Infrared Imaging System
Western blot protocol including example buffers and reprobing procedure
Image Studio Lite Western Blot Analysis Software Western Blotting Principles and Methods Handbook