Immunoblotting from immobilized pH gradients. The case of alpha 2-macroglobulin.
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).
Gelfi
C
C
Chair of Biochemistry, Faculty of Pharmacy, University of Milan, Italy.
Righetti
P G
PG
Cattò
N
N
Bontempi
L
L
Gianazza
E
E
D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007525:Isoelectric Focusing / Q000379:methods*; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds; D014508:Urea; D000511:alpha-Macroglobulins / Q000032:analysis*
D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; D014508:Urea