Adsorption, immunoadsorption and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of proteins using nitrocellulose membrane filters

Abstract

AbstractA rapid analytical procedure is described to perform sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of proteins specifically adsorbed on antibody‐coupled nitrocellulose membrane filters. Purified antibodies were adsorbed on nitrocellulose filters and were immobilized by treatment with dilute glutaraldehyde. Efficient adsorption of specific antigen occurred upon filtration of the antigen solution (e. g. cell supernatant or cell lysate) within a few minutes. Theim‐munoadsorbed proteins were released from the membrane filters in the presence of urea, Nonidet P‐40 detergent and electrophoresis sample buffer and were directly analyzed by SDS‐PAGE. All adsorptions and washing steps were performed by filtration utilizing a filtration manifold. This allowed the processing of a large number of samples in a short time. Furthermore, and in contrast to the Western blotting method, antigen‐antibody recognition preceeded gel electrophoresis, which greatly reduced potential failures in antigen recognition. Similarly, SDS‐PAGE of proteins non‐specifically adsorbed on nitrocellulose filters was also possible. Dilute protein solutions, like cell supernatants, were filtered through nitrocellulose membrane filters. Hereby proteins, but not low molecular weight compounds such as inorganic salts, amino acids or detergents, were rapidly and quantitatively adsorbed. The adsorbed proteins were released from the filters and subjected to SDS‐PAGE as in the previous method.