ABSTRACT
Immunoblotting is a very useful and relatively simple technique used in many fields of medical research to determine the presence, quantity and molecular weight of solubilized proteins, and for assaying the quality and specificity of antibodies. Major requirements for the technique are electrophoresis and immunoblotting apparatus, labelled secondary antibody and antibodies which recognize the proteins of interest. The most common method of gel electrophoresis used when immunoblotting is Tris/glycine sodium dodecyl sulphate polyacrylamide gel electrophoresis. Proteins are transferred from the gel to a membrane support by electrophoretic transfer in order to carry out immunolabelling. Cross-reactivity of the primary antibody with proteins other than the protein of interest may be a problem and can sometimes be alleviated using lower antibody concentrations. Immunoblotting is commonly used in neuroscience to assess the presence of particular proteins, to measure the relative amounts of proteins in different tissues or samples and to detect tyrosine phosphorylation of proteins, using phosphotyrosine antibodies.
