Western blot protocol is the exact standards by which the immunoblot test is conducted. Western blot is used to detect proteins in a tissue sample. The process separates native proteins by determining the lengths of 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 gathered either from living tissue or from a cell culture. Tissue is broken down and various inhibitors such as protease or phosphatase are introduced to prevent enzymes from performing digestion. This portion of the protocol is usually handled in cold temperatures to preserve the tissues.
The gel electrophoresis process is the next step in 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 buffer of sodium dodecyl sulfate. Basically, the proteins become negatively charged and move towards a positively charged electrode within the gel.
Transfer of the proteins is the next part of the overall western blot process. A membrane is placed on top of the gel, followed by filter paper. As an electric current is administered, proteins are pulled into the membrane. This is referred to as the actual “blotting” portion of the analysis. The membranes are highly fragile and easily susceptible to damage.
The correct flow of the western blot protocol calls for the step known as binding. In order to prevent contamination of the proteins themselves by the antibodies that need to be added, a sort of shielding needs to be implemented. The most common type of blocking uses non-fat dry milk and a detergent. This binds to the open spots in the membrane and allows for clearer results when the antibody is added.
Detection is the next step according to correct protocol. The proteins are introduced to antibodies which have been linked to a specific enzyme which will give researchers the desired information. The antibody is incubated with the membrane for 30 minutes or more. Afterward, the membrane is washed and a secondary antibody is introduced that binds to the first. Oftentimes, a luminescent agent is used to assist scientists with the identification process.
The material is washed again to remove any unbound items. Analysis of the size of the stained bands reveals information regarding the prominence and extent of a specific protein. This is generally completed a few times to ensure of proper analysis. Options for detection include x-rays, coloring and chemicals.