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  • Title: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and centrifuge blotting: preparation of polypeptides for amino-terminal sequence analysis.
    Author: Hermansen LF, Juul J, Sletten K.
    Journal: Electrophoresis; 1996 Apr; 17(4):720-2. PubMed ID: 8738334.
    Abstract:
    The applicability and reproducibility of a previously described (L. F. Hermansen et al., Electrophoresis 1993, 14, 1302-1306) centrifuge-blotting procedure for capturing subnanomolar amounts of protein on polyvinylidene difluoride membranes for direct Edman degradation was further investigated. Proteins with different molecular masses were centrifuge-blotted onto Immobilon CD membranes. Simultaneous blotting and desalting was achieved with an overall yield of 15-56% after 2 h centrifugation for proteins with a molecular mass of 12-30 kDa. Centrifugation of myoglobin for 6 h resulted in an overall yield of 72%. The subnanomolar amounts obtained were also sufficient to conduct cyanogen bromide cleavage in situ on proteins with blocked NH2-terminus and to generate sequence information.
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