IMMUNOBLOTTING FROM IMMOBILIZED PH GRADIENTS - THE CASE OF ALPHA-2-MACROGLOBULIN

Guidelines for effective blotting of preteins from immobilized pH gradients with a soft polyacrylamide matrix (e.g. T% = 3) include: thick (1 mm) IPG slabs, electrotransfer in a buffer tank in the presence of 0.1% SDS, nitrocellulose of the sturdiest type, thorough removal of all IPG fragments before further processing of the membrane. For α2-M, IPG on a 4-6.5 gradient followed by enzyme-linked immunodetection allows the recognition oof a complex pattern with several bands centered around pI 5.1. The procedure may also reveal the desialylated forms of α2-M (microheterogeneity reduced to 2-3 bands), the native subunits (after reduction with thiols) and the denatured half molecules (in the presence of 8 M urea).