Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

TLDR

The procedure is intended for analyzing diverse proteins using specific reactions or ligands. Proteins are transferred to nitrocellulose, blocked with excess protein, then probed with a primary antibody followed by a labeled secondary antibody for detection by autoradiography, fluorescence, or peroxidase. The method achieves quantitative transfer of ribosomal proteins from urea gels, preserves band patterns in SDS gels (though not quantitatively), and enables detection of as little as 100 pg by autoradiography, fluorescence, or peroxidase, facilitating immunological assays.

Abstract

A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.