Quantification of Protein in Brain Tissue by Western Immunoblot Analysis

Western blots are designed to determine protein levels and their patterns of modification in homogenized tissue samples. Western blots are quantifiable, but unlike immunohistochemistry, the cellular integrity is lost. Both Western blots and immunohistochemistry depend on the availability of antibodies against the protein of interest. Antibodies may be directed not only against a protein, but can also be directed against a chemical modification of the protein, such as phosphorylation of specific amino acid residues or glycosylation. For Western blots, the proteins in the sample are denatured, size-separated on a denaturing acrylamide gel, and transferred to a nylon membrane. An antibody is reacted with the proteins on the membrane and binds to its specific antigen. The resulting antibody-antigen complex is visualized with the help of a chemiluminescent assay system that darkens X-ray films.