Immunoblot affinity purification

The immunoblotting method has evolved from early stages when antibodies were used to 'stain' polyacrylamide gels directly^1,2 to more versatile methods using replica techniques, in which the separated polypeptides are transferred to nitrocellulose membranes, chemically activated paper or nylon sheets. Although there are several variations on this basic theme, the most common and effective is electrophoretic transfer to nitrocellulose sheets^3,4. The separated proteins can then be probed with antibodies; this variation of the technique is known as immunoblotting (or western blotting). The membrane can also be probed with specific ligands, such as DNA, protein, small molecules (for example, heparin or GTP) or even whole cells. Electrophoretic transfer can be achieved either in a tank³ or in a semidry apparatus⁵, in which the buffer volume is reduced to filter paper pads. The following protocol presents the original method used for tank blotting.