Centrifuge‐blotting of proteins after separation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

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 centrifugeblotting procedure involves the following steps: (i) visualization of the protein in a sodium dodecyl sulfate (SDS)‐polyacrylamide gel with 1 M KC1, (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.