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).
IMMUNOBLOTTING FROM IMMOBILIZED PH GRADIENTS - THE CASE OF ALPHA-2-MACROGLOBULIN / C. GELFI, P. RIGHETTI, N. CATTO, L. BONTEMPI, E. GIANAZZA. - In: JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS. - ISSN 0165-022X. - 16:2-3(1988), pp. 193-204.
IMMUNOBLOTTING FROM IMMOBILIZED PH GRADIENTS - THE CASE OF ALPHA-2-MACROGLOBULIN
C. GELFIPrimo
;E. GIANAZZAUltimo
1988
Abstract
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).Pubblicazioni consigliate
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