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The Western blot protocol is used to detect proteins in a tissue sample. It involves tissue collection, gel electrophoresis, protein transfer, binding, and detection. The process separates native proteins by determining the lengths of the polypeptides using a gel electrophoresis. The proteins themselves are then transferred to a nitrocellulose membrane and probed by antibodies. The detection options include x-rays, dyes, and chemicals.
The Western blot protocol is the exact standard by which immunoblot testing is conducted. Western blot is used to detect proteins in a tissue sample. The process separates native proteins by determining the lengths of the polypeptides using a gel electrophoresis. The proteins themselves are then transferred to a nitrocellulose membrane and probed by antibodies.
The first part of the western blot protocol begins with tissue collection. These samples are collected from living tissue or a cell culture. The tissue is broken down and various inhibitors such as protease or phosphatase are introduced to stop the enzymes from carrying out the digestion. This part of the protocol is usually run at low temperatures to preserve tissue.
The gel electrophoresis process is the next step in the western blot protocol. In this portion the proteins are identified by various factors such as molecular weight or electrical charge. This process is most commonly completed using polyacrylamide gels with a sodium dodecyl sulfate buffer. Basically, the proteins become negatively charged and move towards a positively charged electrode within the gel.
Protein transfer is the next part of the whole western blot process. A membrane is placed over the gel, followed by filter paper. When an electric current is given, the proteins are pulled into the membrane. This is referred to as the effective “blotting” portion of the analysis. The membranes are highly fragile and easily susceptible to damage.
Proper flow of the Western blot protocol requires the step known as binding. In order to prevent contamination of the proteins themselves by the antibodies to be added, some sort of shielding must be implemented. The most common type of blockage uses powdered skim milk and a detergent. This binds to open spots in the membrane and allows for clearer results when the antibody is added.
Detection is the next step based on the correct protocol. The proteins are introduced into antibodies that have been linked to a specific enzyme that will provide the researchers with the desired information. The antibody is incubated with the membrane for 30 minutes or more. Subsequently, the membrane is washed and a secondary antibody is introduced which binds to the first. Often, a luminescent agent is used to assist scientists in the identification process.
The material is washed again to remove any unbound elements. Analysis of the size of the colored bands reveals information regarding the prominence and extent of a specific protein. This is generally completed a few times to ensure proper parsing. Detection options include x-rays, dyes, and chemicals.
