Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the discontinuous system of Laemmli is used world-wide for analytical and preparative gel electrophoresis of polypeptides. A minor but disturbing problem is the difficulty of concentrating highly diluted solutions and determining their protein content after 2-mercaptoethanol-SDS solubilization. We describe a solution to both of these problems, detailing a two-step procedure which takes advantage of the low solubility of potassium dodecyl sulfate (KDS). Removal of excess of SDS and 2-mercaptoethanol, and concentration of proteins from even a nanomolar solution, is achieved by a two-step KDS precipitation. Free dodecyl sulfate is precipitated in step one, while KDS-proteins are pelleted in the second step, allowing the thiol agents to be discarded with the supernatant. The effects of changing [SDS] and [KC1], temperature, and pH were studied to optimize the separation of free SDS from proteins. After final precipitation, the hundred- or thousandfold concentrated proteins can be suspended in a small volume of any required medium. The procedure allows protein determination by the Lowry method, peptide mapping of 2-mercaptoethanol-SDS-solubilized polypeptides, and all other analyses which are otherwise hampered by excesses of SDS and/or thiol reagents.
Sandri
M
M
Department of Experimental Biomedical Sciences, University of Padua, Italy.
Rizzi
C
C
Catani
C
C
Carraro
U
U
D000818:Animals; D011232:Chemical Precipitation; D002623:Chemistry Techniques, Analytical / Q000379:methods; D007851:Dodecanol; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000302:isolation & purification*; D011188:Potassium; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D012967:Sodium Dodecyl Sulfate / Q000302:isolation & purification*; D012995:Solubility; D013696:Temperature
D011506:Proteins; D007851:Dodecanol; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D011188:Potassium