Western blotting of basic proteins after nondenaturing electrophoresis in acid conditions using the PhastSystem.
Electroblotting of basic proteins was performed from minigels after electrophoresis, under nondenaturing acidic conditions, by using the automated PhastSystem. Depending on the molecular masses of the proteins to be studied, various precast gel media were chosen. The transfer membranes with various types (nitrocellulose and polyvinylidene difluoride) and pore sizes (0.45 and 0.2 micron) were chosen accordingly. For the semidry electric transfer, a simple, discontinuous two-buffer system was used. The anode solution contained 0.3 M Tris, pH 10.4, and the cathode solution, 40 mM 6-amino-n-hexanoic acid, pH 7.6, with 20% v/v methanol each. The addition of 0.1% sodium dodecyl sulfate (SDS) in the cathode solution facilitated the elution of proteins from the gels and directed the migration of the negative SDS-protein complexes towards the anode membranes. The transfer conditions following native polyacrylamide gel electrophoresis allowed the visualization of basic proteins, with molecular weights ranging from 29,000 to 5,000, for which isoforms could be resolved and which retained their biological properties.
Davril
M
M
Unité INSERM N. 16, Lille, France.
Ducourouble
M P
MP
Van-Seuningen
I
I
D015153:Blotting, Western / Q000379:methods*; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D019272:Leukocyte Elastase; D008567:Membranes, Artificial; D009113:Muramidase / Q000032:analysis; D010196:Pancreatic Elastase / Q000032:analysis; D011145:Polyvinyls; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D005465:Fluorocarbon Polymers
D003101:Collodion; D019272:Leukocyte Elastase; D008567:Membranes, Artificial; D009113:Muramidase; D010196:Pancreatic Elastase; D011145:Polyvinyls; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers