Anal Biochem. 1993 Aug 15;213(1):34-9. Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation. Sandri M, Rizzi C, Catani C, Carraro U. Source Department of Experimental Biomedical Sciences, University of Padua, Italy. Abstract 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. PMID: 8238879 [PubMed - indexed for MEDLINE]
SELECTIVE REMOVAL OF FREE DODECYL-SULFATE FROM 2- MERCAPTOETHANOL SDS-SOLUBILIZED PROTEINS BEFORE KDS PROTEIN PRECIPITATION
SANDRI, MARCO;CARRARO, UGO
1993
Abstract
Anal Biochem. 1993 Aug 15;213(1):34-9. Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation. Sandri M, Rizzi C, Catani C, Carraro U. Source Department of Experimental Biomedical Sciences, University of Padua, Italy. Abstract 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. PMID: 8238879 [PubMed - indexed for MEDLINE]
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