23 - Immunoblotting techniques

Abstract

This chapter provides an overview of the blotting techniques that are used to study a macromolecule with respect to size and/or isoelectric point and other specific characters. As the molecular weights of proteins are on average much lower than those of nucleic acids, their separation by electrophoresis is carried out in polyacrylamide gels, which allow very poor diffusion of macromolecules off the gel. To overcome this difficulty, two approaches are developed almost simultaneously. In the first approach, composite polyacrylamide/agarose gels are prepared with a reversible crosslink to allow diffusion by standard blotting techniques after degrading the acrylamide matrix. In the second approach, proteins are transferred off the gel by electrophoresis. Diazo paper is used in the first approach, whereas nitrocellulose membranes are used in the second. Protein blotting techniques are currently known as “Western” blotting, a name derived from the previously coined “Northern” blots versus “Southem” blots. Whereas the Northern and Southern blotting techniques detect specific ribonuclic acid (RNA) or deoxy-ribonuclic acid (DNA) sequences by hybridization with radioactive or nonradioactive probes, Western blotting generally detects the presence of different proteins by incubating the membrane with specific antibodies directed against them.