Centrifuge-blotting of proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
| Autor: | Leonila F. Hermansen, Knut Sletten, Öyvind Pedersen |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Clinical Biochemistry
Centrifugation Biochemistry Potassium Chloride Analytical Chemistry Dialysis tubing chemistry.chemical_compound Sodium dodecyl sulfate Polyacrylamide gel electrophoresis Gel electrophoresis Chromatography Myoglobin Chemistry Methanol Proteins Membranes Artificial Serum Albumin Bovine Chymotrypsinogen Molecular Weight Electrophoresis Membrane Distilled water Electrophoresis Polyacrylamide Gel Polyvinyls Sequence Analysis |
| Zdroj: | Electrophoresis. 14:1302-1306 |
| ISSN: | 1522-2683 0173-0835 |
| DOI: | 10.1002/elps.11501401199 |
| Popis: | A protein transfer method which allows elution and immobilization of polypeptides onto a polyvinylidene difluoride (PVDF) membrane has been developed. The protein band in a gel is eluted by centrifugation. The centrifuge-blotting procedure involves the following steps: (i) visualization of the protein in a sodium dodecyl sulfate (SDS)-polyacrylamide gel with 1 M KCl, (ii) excision of the protein band and equilibration for 15 min in a solution of 0.05% SDS/5% methanol/0.02% dithiothreitol in distilled water, (iii) placing the gel piece in direct contact with the PVDF membrane in the receptacle, (iv) centrifugation at 3000 g for 1 h. A 10 kDa cut-off dialysis membrane is placed beneath the PVDF membrane to retain nonimmobilized protein. The N-terminal sequence of the immobilized protein on the PVDF membrane was determined. For proteins with a molecular mass less than 30 kDa, an overall yield between 10%-30% has been obtained. |
| Databáze: | OpenAIRE |
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