Isoelectric focusing of immunoglobulins: improved methodology

Conditions for isoelectric focusing and detecting antibodies in thin layers of polyacrylamide have been evaluated and several improvements have been made. First, we have developed a simple method for covalently attaching the polyacrylamide gel to the glass support which improves the mechanical stability of the gel and removes the need for protein subbed plates. This in turn leads to decreased electroendosmosis and decreased background protein staining. Secondly, we have applied the methods of Nguyen and Chrambach (1977 a,b), in which amino acid solutions are used as electrodes, to focus immunoglobulins. This has eliminated cathodic drift and resulted in extremely stable pH gradients. Finally, we have found that conventional methods for detecting focused antibodies that rely on protein cross-linking before exposure to antigen often lead to distortion of the focusing patterns. Both glutaraldehyde and suberimidate destroy the antigen binding capacity of some antibodies at concentrations too low for complete fixation of protein bands. These fixation artifacts are avoided if salt-precipitated antibodies are exposed to radiolabeled antigen before fixation.