Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation

M. Sandri, C. Rizzi, C. Catani, U. Carraro

Research output: Contribution to journalArticle

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[KCl], 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.

Original languageEnglish
Pages (from-to)34-39
Number of pages6
JournalAnalytical Biochemistry
Volume213
Issue number1
DOIs
Publication statusPublished - 1993

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Mercaptoethanol
Proteins
Electrophoresis
Peptides
Sulfhydryl Reagents
Peptide Mapping
Sulfhydryl Compounds
Sodium Dodecyl Sulfate
Solubility
dodecyl sulfate
Polyacrylamide Gel Electrophoresis
Gels
Temperature

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation. / Sandri, M.; Rizzi, C.; Catani, C.; Carraro, U.

In: Analytical Biochemistry, Vol. 213, No. 1, 1993, p. 34-39.

Research output: Contribution to journalArticle

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