Quantitation of proteins separated in N, N'-1,2-dihydroxyethylenebisacrylamide-crosslinked polyacrylamide gels

Anal Biochem. 1992 Oct;206(1):1-5. doi: 10.1016/s0003-2697(05)80002-8.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acrylamides*
  • Acrylic Resins
  • Animals
  • Cattle
  • Cross-Linking Reagents
  • Electrophoresis, Polyacrylamide Gel / methods*
  • Proteins / isolation & purification*
  • Rosaniline Dyes
  • Serum Albumin, Bovine / isolation & purification
  • Sodium Dodecyl Sulfate

Substances

  • Acrylamides
  • Acrylic Resins
  • Cross-Linking Reagents
  • Proteins
  • Rosaniline Dyes
  • polyacrylamide gels
  • Serum Albumin, Bovine
  • Sodium Dodecyl Sulfate
  • N,N'-(1,2-dihydroxyethylene)bisacrylamide
  • coomassie Brilliant Blue