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  • Title: Centrifuge-blotting of proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
    Author: Hermansen LF, Pedersen O, Sletten K.
    Journal: Electrophoresis; 1993 Dec; 14(12):1302-6. PubMed ID: 8137793.
    Abstract:
    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.
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