7.25D: Western Bolts
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- Show the uses of Western Blots
The Western blot (sometimes called the protein immunoblot) is a widely accepted analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Western blot samples can be taken from whole tissue or from cell culture. Solid tissues are first broken down mechanically using either a blender (for larger sample volumes), a homogenizer (smaller volumes), or by sonication. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. The technique 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 (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein. There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against tens of thousands of different proteins belonging to signaling pathways or cell surface receptor antigens, or other cellular or soluble components. 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 using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA). This method originated in the laboratory of George Stark at Stanford. 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.
- After separation by gel electrophoresis using SDS-PAGE, proteins are transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel.
- The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody is conjugated to alkaline phosphatase or horseradish peroxidase.
- The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.
- electrophoresis: a method for the separation and analysis of large molecules (such as proteins) by migrating a colloidal solution of them through a gel; gel electrophoresis