title: Immunoblotting from immobilized pH gradients. The case of alpha 2-macroglobulin.
abstract: Guidelines for effective blotting of proteins 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 alpha 2-M, IPG on a 4-6.5 gradient followed by enzyme-linked immunodetection allows the recognition of a complex pattern with several bands centered around pI 5.1. The procedure may also reveal the desialylated forms of alpha 2-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).
authors: Chair of Biochemistry, Faculty of Pharmacy, University of Milan, Italy.
authors: C
authors: C
authors: Gelfi
authors: P G
authors: PG
authors: Righetti
authors: N
authors: N
authors: Cattò
authors: L
authors: L
authors: Bontempi
authors: E
authors: E
authors: Gianazza
mesh_terms: D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007525:Isoelectric Focusing / Q000379:methods*; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds; D014508:Urea; D000511:alpha-Macroglobulins / Q000032:analysis*
chemical_list: D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; D014508:Urea