Western Blotting Overview
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Introduction
The western blot (immunoblot) technique is the most common method for protein detection in molecular biology. Used all over the world, the immunoblotting utilizes the high affinity relationship between antibodies and antigens to detect proteins from cellular lysates, tissues, or other extracts.
As antibodies have improved, western blots have been used for the diagnosis of several diseases including HIV and lyme disease. This article will offer a broad overview of the western blot process, as well as a discussion of its application in biomedical diagnostics.
There are basically six steps to a western blot protocol:
- Sample preparation (lysis and denaturing).
- Running the gel (resolving the proteins by mass size)
- Transferring to a membrane
- Blocking the membrane
- Antibody incubation
- Film development
Because of its relative simplicity, the immunoblot is the most popular technique for protein detection in molecular biology. The assay can be completed in one day (if the antibodies are good) and allows for relative comparisons of protein abundance between samples.
To start, proteins are dissolved in a buffer with a small amount of detergent. For conditions in which the proteins are denatured, a detergent called sodium dodecyl sulfate (SDS) is used. The addition of a reducing agent, usually beta-mercaptoethanol, is also used to break disulfide bonds and fully denature the protein from its native state.
After proteins are denatured, they are resolved on a polyacrylamide gel by molecular weight. Next, the proteins are transferred to a membrane (nitrocellulose or PVDF) with a high protein binding capacity. Membranes are then blocked with one of a variety of protein blocking buffers. Blocked membranes are then incubated with a primary antibody that recognizes a specific cellular protein. An enzyme conjugated secondary antibody then detects the primary antibody. The membrane is then exposed to a film in the presence of a chemiluminescent substrate to detect the amount of proteins on the membrane.
Sample Preparations
Eukaryotic cells are packed with proteins which are folded into tight, three-dimensional shapes. In order to perform a western blot, most people denature cellular proteins. In other words, proteins are solublized with detergents and intramolecular disulfide bonds are broken to reduce proteins into long chains of amino acids. A typical lysis buffer, called Laemlli buffer, is composed as follows:
Laemlli Buffer
- 50 mM Tris-HCl pH 6.8
- 2% SDS
- .0025% Bromophenol Blue (pH-sensitive dye)
- 10% Glycerol
- 5% Beta-mercaptoethanol
This buffer can be added directly to cells or tissues to solublize the proteins. The beta-mercaptoethanol gives the buffer a very strong odor, and is also considered a toxic molecule, so it should be handled with care. Once the Laemmli buffer is added, samples are boiled for 5 minutes to completely denature the proteins.
Courtesy of jepoirrier on Flickr CC
Polyacrylamide Gels and Protein Resolution
Protein samples are resolved on a polyacrylamide gel using an electrical current (called electrophoresis). To achieve a compact protein band, two different phases of polyacrylamide gel electrophoresis (PAGE) are used: a stacking gel on top, and a resolving gel on the bottom.
The Stacking Gel
The stacking gel is a low percentage polyacrylamide gel. As proteins migrate through a polyacrylamide matrix, they make contact with the gel and create friction. However, the force of friction between proteins and polyacrylamide is negligible in a low-percentage stacking gel. Consequently, the migration of proteins is unrestrained by molecular weight. However, a wall of chloride ions from the buffer of the gel limits the electrophoretic mobility of proteins being resolved in the stacking gel. The proteins are therefore compacted into a tight band, limited at the lower end by the fixed migration of chloride ions.
The force of friction is negligible within the stacking gel because the polyacrylamide percentage is low. The protein mobility in the stacking gel is limited by a wall of chloride ions, which slows the migration of proteins until the lysate is concentrated into a sharp band. © HowtoWesternBlot.net
The Resolving Gel
The bottom portion of a polyacrylamide gel is the resolving gel. Generally, the resolving gel is between 6-15% polyacrylamide. The increased percentage of polyacrylamide in the resolving gel creates an increased force of friction as the proteins migrate through the matrix. At the stacking/resolving gel interface, the force of friction changes from negligible to dominant, and proteins begin to be resolved by molecular weight: smaller proteins migrate faster, and larger proteins migrate slower. This element of size separation adds an additional layer of stringency to the western blot technique. Not only does an antibody have to detect the protein of interest, but the protein must also be at the correct molecular weight.
When the proteins reach the stacking/resolving interface, the force of friction becomes dominant and proteins are resolved by molecular weight. © HowtoWesternBlot.net
Membrane Transfer
After the proteins have been resolved on a polyacrylamide gel, they are transferred to a thin membrane. This membrane is usually composed of nitrocellulose or a material called polyvinylidene fluoride (PVDF), and has a large binding capacity for proteins.
The protein-containing gel is sandwiched between buffer-soaked filter papers in a machine capable of producing an electrical current. The membrane is placed immediately next to the gel, on the anode side of the machine.
A traditional Transfer Buffer is composed of the following:
- 50 mM Tris Base
- 40 mM Glycine
- 20% Methanol
As a current passes through the gel, proteins migrate out of the gel and bind to the membrane (see figure). Consequently, the protein resolution of the gel is preserved, but the proteins are now bound to a membrane which can be probed with various antibodies to detect specific proteins.
Western Blot Transfer
Negatively charged proteins move out of the gel toward the anode of the transfer apparatus. As the proteins migrate out of the gel, they bind to the membrane (nitrocellulose or PVDF) which is immediately under the gel. Source: bensaccount on Wikimedia Commons
Blocking Western Blot Membranes
Before membranes can be probed with antibodies, they need to be “blocked” with one of a variety of blocking agents. The enormous number of cellular proteins raises the potential for western blot antibodies to react non-specifically with cellular proteins. Non-specific detection prevents researchers from detecting the protein of interest, and the assay fails.
A variety of blocking agents are used to solve this problem. Common blocking agents include:
- Bovine serum albumin
- Non-fat milk
- Fish Gelatin
- Serum
- Protein-free blocking buffers
Each of these blocking buffers confers different advantages, and a technical discussion of each agent’s positive and negative features is beyond the scope of this article. However, there is plenty of information available in our western blot blocking buffers page, if you need to reference it.
Antibody Incubation
Once the membrane is blocked, a primary antibody is added to detect a specific protein of interest. Antibodies are commercially available from a large number of companies. The primary antibody directly binds to the protein of interest, but is engineered to be specific enough that it won’t react with other cellular proteins.
After primary antibody binding, a secondary antibody is added to detect the primary antibody. Usually, the secondary antibody is conjugated to an enzyme called horseradish peroxidase (HRP). After incubating the western blot membrane with secondary antibody, a series of wash steps clears away the antibodies and prepares the membrane for chemiluminescent detection.
Development
The most common method of western blot detection uses film development. At this point, cellular proteins are bound to a membrane, primary antibody is bound to a protein of interest, and secondary antibody is bound to the primary antibody.
The secondary antibody is conjugated to HRP because HRP will produce light when exposed to the correct substrate. This substrate is called luminol, and HRP catalyzes the conversion of luminol to 3-aminophthalate in a reaction that releases light. The light can be detected on a film, and the amount of emitted light is used as a correlate to the amount of protein on the membrane. In the example on the right, one can conclude that the lane on the far right contains less protein of interest than the other lanes.
Diagnostic Applications
Due to its ability to detect proteins from tissues and cells, the western blot can be used to diagnose medical conditions based on the expression pathogenic proteins.
Western Blot Test for HIV
HIV can be diagnosed by western blotting for viral proteins from human blood samples. This test is usually used in combination with another form of immunodetection called an enzyme linked immunosorbent assay (ELISA).
Additionally, mad cow disease (bovine spongiform encephalopathy) can be confirmed by western blot detection of the prion protein PrPSc. In this case, I write “detected” rather than “diagnosed” because the test must be performed on post-mortem brain tissue to confirm mad cow disease.
Finally, the immunoblots and ELISA combination are the most common method for diagnosing Lyme disease.
Conclusion
The western blot has been used for years as the most common method of protein detection in molecular biology. As antibodies for immunodetection have improved, western blotting has found a place in medical diagnostics to detect the presence of pathogenic proteins. Modern improvements are currently taking place to reduce the time of the assay, as well as add digital imaging options instead of film. Even as other methods of protein detection become available, it seems unlikely that the classic western blot will be replaced because of its reliability, simplicity, and the widespread availability of antibodies for immunodetection.
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