Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation.
Anal Biochem
; 213(1): 34-9, 1993 Aug 15.
Article
en En
| MEDLINE
| ID: mdl-8238879
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Dodecil Sulfato de Sodio
/
Proteínas
/
Mercaptoetanol
Límite:
Animals
Idioma:
En
Revista:
Anal Biochem
Año:
1993
Tipo del documento:
Article
País de afiliación:
Italia
Pais de publicación:
Estados Unidos