Abstract
Following separation of proteins by SDS-PAGE, they are electroblotted onto polyvinylidene difluoride membranes (Immobilon). Protein bands of interest are excised, and the proteins are eluted from the membrane with detergent-containing buffers at pH 9.5. The method routinely yields recovery of 70–90%, and this is independent of protein molecular weight.
Similar content being viewed by others
References
Tuszynski, N. Y., Damsky, C. H., Fuhrer, J. P., and Warren, L. (1977) Recovery of concentrated protein samples from sodium dodecyl sulfate-polyacrylamide gels.Anal. Biochem. 83, 119–129.
Nguyen, N. Y., DiFonzo, J., and Chrambach, A. (1980) Protein recovery from gel slices by steady-state stacking: An apparatus for the simultaneous extraction and concentration of ten samples.Anal. Biochem. 106, 78–91.
Hager, D. A. and Burgess, R. R. (1980) Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: Results with sigma subunit ofEscherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes.Anal. Biochem. 109, 76–86.
Stralfors, P. and Belfrage, P. (1983) Electrophoretic elution of proteins from polyacrylamide gel slices.Anal. Biochem. 128, 7–10.
Hunkapiller, M. W., Lujan, E., Ostrander, F., and Hood, L. E. (1983) Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis.Methods Enzymol. 91, 227–236.
Parekh, B. S., Mehta, H. B., West, M. D., and Montelaro, R. C. (1985) Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Anal. Biochem. 148, 87–92.
Anderson, P. J. (1985) The recovery of nitrocellulose-bound protein.Anal. Biochem. 148, 105–110.
Szewczyk, B. and Summers, D. F. (1988). Preparative elution of proteins blotted to immobilon membranes.Anal. Biochem. 168, 48–53.
Szewczyk, B., Laver, W. G., and Summers, D. F. (1988) Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase.Proc. Natl. Acad. Sci. USA 85, 7907–7911.
Manson, M. (ed.) (1992)Methods in Molecular Biology, vol. 10: Immunochemical Protocols, Humana, Totowa, NJ.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Szewczyk, B., Summers, D.F. Efficient elution of purified proteins from polyvinylidene difluoride membranes (immobilon) after transfer from SDS-PAGE and their use as immunogenes. Mol Biotechnol 2, 129–134 (1994). https://doi.org/10.1007/BF02824805
Issue Date:
DOI: https://doi.org/10.1007/BF02824805