Quantitation of proteins separated in N, N'-1,2-dihydroxyethylenebisacrylamide-crosslinked polyacrylamide gels.
A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N, N'-1, 2-dihydroxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 micrograms and not disturbed by dissolved gel. Separations in N, N'-1, 2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels.
Neumann
U
U
Institut für Medizinische Chemie, Medizinische Hochschule Hannover, Germany.
Khalaf
H
H
Rimpler
M
M
D000178:Acrylamides; D000180:Acrylic Resins; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification*; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
D000178:Acrylamides; D000180:Acrylic Resins; D003432:Cross-Linking Reagents; D011506:Proteins; D012394:Rosaniline Dyes; C016680:polyacrylamide gels; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; C071115:N,N'-(1,2-dihydroxyethylene)bisacrylamide; C004692:coomassie Brilliant Blue