How to Run a Western Blot
The Western blot is a general technique in analytical biochemistry for detecting specific proteins in a tissue sample. It uses gel electrophoresis to physically separate the proteins according to criteria such as size or structure. The proteins with the physical characteristics of the target protein may then be probed with antibodies that are specific to the target protein. The samples are then treated to make these antibodies detectable.
Things You'll Need
- Blender
- Electrophoresis gel
- Enzymes
- Filter paper
- Nitrocellulose membrane
- Protein sample
- Substrate
Instructions
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Take a sample from whole tissue or a tissue culture. Break down larger pieces with a blender or some other mechanical method. You can also use the Western blot on other sources of protein such as bacteria, viruses and environmental samples.
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2
Separate the proteins in the sample with gel electrophoresis. Place the protein samples in a special gel and separate them according to some criteria such as electrical charge or molecular mass. For example, you can separate the proteins by applying an electrical charge on one end of the gel plate. The smaller proteins will then move more quickly toward the charge.
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3
Transfer the proteins in the gel onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Place the membrane on top of the gel and weigh it down with a stack of filter paper. The proteins will move to the membrane through capillary action.
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Block any potential interaction between the membrane and the antibodies that you will add later. Place the membrane in a diluted solution of protein. This protein will attach to any places on the membrane where the target protein is not already attached.
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Load the membrane with specific antibodies. These antibodies will attach only to the proteins of specific interest. Expose the membrane to an enzyme and then a substrate. This will cause some type of visible reaction, such as a color change, to indicate the location of the target proteins.
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