Neumann U, Khalaf H, Rimpler M
Institut für Medizinische Chemie, Medizinische Hochschule Hannover, Germany.
Anal Biochem. 1992 Oct;206(1):1-5. doi: 10.1016/s0003-2697(05)80002-8.
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.
本文介绍了一种简单快速的方法，用于定量通过十二烷基硫酸钠电泳或平板凝胶等电聚焦分离的蛋白质。该方法基于用N，N'-1,2 - 二羟基亚乙基双丙烯酰胺（DHEBA）交联的聚丙烯酰胺凝胶在高碘酸中的溶解性。电泳分离后，蛋白质用考马斯亮蓝G - 250染色。DHEBA凝胶在染色和脱色过程中会显著膨胀，但在10%（w/v）硫酸铵溶液中可收缩至正常大小。从凝胶上切下染色条带并在高碘酸中溶解。溶解过程中染料会褪色。溶液中的蛋白质浓度通过改良的考马斯染料结合测定法确定。定量范围在100 ng至5 μg之间呈线性，且不受溶解凝胶的干扰。在N，N'-1,2 - 二羟基亚乙基双丙烯酰胺交联凝胶中的分离显示出与正常交联凝胶相似的性质。
