A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances.
A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.
Guttenberger
M
M
Universität Tübingen, Institut für Botanik, Federal Republic of Germany.
Neuhoff
V
V
Hampp
R
R
D000545:Amanita; D000665:Ampholyte Mixtures; D002021:Buffers; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000032:analysis; D015151:Immunoblotting / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D008722:Methods; D009693:Nucleic Acid Hybridization; D012044:Regression Analysis
D000665:Ampholyte Mixtures; D002021:Buffers; D005656:Fungal Proteins; D008565:Membrane Proteins