The western blotting technique was developed in 1979 by Towbin. The system received its name from the step where a protein is “blotted” from a gel onto a membrane and as a spinoff from the name of the southern blot technique. The western blot has become a routine technique in the detection of target proteins in a mixture. The western blot produces both qualitative and semi quantitative data about the protein in question.
One of the first steps in western blotting is sample preparation. Samples from tissues or cells are broken using common lysis techniques such as a blender, homogenizer, or a sonicator. Once the tissues are blended in a mix, different detergents such as salts and buffers are used to lyse the cells and solubilize the proteins. A mixture of molecular biology techniques are employed to separate different cell compartments.
The second step in the western blot technique is to perform a gel electrophoresis separation of the proteins in the sample. Proteins can be separated by their isoelectric value (PI), molecular weight, electric charge, or all three. The type of separation performed depends heavily on the type of gel being used such as SDS-PAGE, polyacrylamide, or even acrylamide.
The most widely used gel in western blots is the polyacrylamide gel. The gel is used with a buffer called sodium dodecyl sulfate which keeps the polypeptides in its denatured state. In its denatured state, a linear poplypeptide can travel through a gel pore and be separated by size. Smaller proteins migrate through the acrylamide gel complex faster and larger proteins move slower. The percentage of acrylamide used in gels determines its resolution. For large molecular weights a smaller percentage of acrylamide is used while for small molecular weights a larger percentage of acrylamide is employed. Samples are loaded into wells in a gel which make up lanes. One lane has a ladder which is simply a known standard of proteins of known weights. One electricity is applied the linear proteins migrate to the cathode at a rate that is based on their isoelectric point charge and mass.
The third step is known as the blotting step. In order to allow the proteins to be accessible to antibody detection they must be transferred from a gel to a membrane made of nitrocellulose or polyvinylidene difluoride. The gel is placed on top of a nitrocellulose membrane and then sandwiched between two filter papers. This entire stack is placed into a buffer solution inside an electroblotting box. The solution moves up the paper by capillary action and electric current taking the protein along with it. The protein binds to the nitrocellulose membrane based upon hydrophobic interactions as well as its charges.
The fourth step is known as the blocking step. This step is important in order to prevent antibodies used to detect the protein of interest from non-specific binding interactions with the membrane itself. Blocking is carried out using BSA or dry milk in TBS with a small percentage of a detergent such as Tween 20 or Triton X-100. When the membrane is placed in this solution the blocking protein detergent mix fills in all the spaces on the membrane where no protein is attached from the blotting step. Now when the antibody is added there is no place on the membrane for the antibody to attach. Therefore the antibody only attaches to the protein it recognizes. This leads to more accurate results by removing the chances for false positives.
The next step is to detect the protein of interest using a modified antibody. The antibody is usually linked to some kind of reporter molecule. When this modified antibody is allowed to react with a specific substrate that the enzyme will convert the substrate and produce a color. Traditionally this takes place in two steps:
- First the primary antibody is added. Primary antibody production is usually generated in a host species such as a rabbit, horse, or even exotic animals like llamas. The antigen is injected into the animal and the animal’s serum is harvested for its antibody. After the blocking step a small amount of primary antibody is mixed in with the membrane under mild shaking. The primary antibody is allowed to bind for 1-8 hours. Different temperatures are employed to affect binding both specific and non-specific.
- After the primary antibody is added, allowed to incubate, and washed from the membrane the second antibody is added. This second antibody is against a portion of a host species specifically the animal used to generate the primary antibody. The secondary antibody is linked to a reporter enzyme such as alkaline phosphatase or HRP. HRP is the most common reporter used. A sheet of photographic film is placed against the membrane and then exposed to light. This reaction forms an image of the antibodies bound to the blot. Another form of detection in western blots are to use a static fluorophore linked antibody or radioactive labeled antibodies.
The fifth step in the western blot technique is to analyze the resulting data. Once the unbound probes are washed away, the western blot can be “developed” to detect the bound proteins of interest. Proteins of interest will not always be visualized as one clean band in a membrane. Sometimes more than one band can be visualized. The size of the protein can be estimated by looking at the lane with the ladder. Sometimes the total protein can be visualized against actin or tubulin in order to correct for errors. There are a few ways to go about visualizing the detected proteins:
- Chemiluminescent detection: This method utilizes an enzyme such as HRP which converts a substrate and causes it to luminesce. A CCD camera is used to take an image of the western blot or photographic film. The image is then analyzed by densitometry and quantifies the result in terms of OD (optical density). This method is one of the first methods to be used for western blot detection.
- Fluorphore detection: These immunoassays require fewer steps because there is no need for a substrate to develop the assay, however special equipment must be used to detect a fluorescent signal. Recently digital imaging has shifted towards infrared regions of detection. Near IR and quantum dots has increased the use of fluorescent probes due to their enhanced sensitivity in western blot analysis.
- Radioactive detection: Radioactive label can also be used in analysis. The membrane is placed against an X-ray film and then allowed to develop. The film beings to show the bands of the protein of interest as dark regions. This type of detection is rarely used due health risks.
To summarize, the western blot is a powerful technique used by many labs in order to analyze a protein in a mixture. Western blotting utilizes a series of steps in order to separate, blot, and detect individual proteins. While the technique itself is not new advances in data analysis continue to improve to yield more accurate results.