The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes.
The determination of the immunoreactivity of protein antigens in complex mixtures has been greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer to nitrocellulose membrane (NCM), and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents have been published, but no studies have been published which compare these methods with each other or with others of potential merit. We have performed such a comparative study using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, we describe a method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins. This system proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. In addition, individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay.
D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001345:Autoradiography; D001426:Bacterial Proteins / Q000276:immunology*; D001667:Binding, Competitive; D002482:Cellulose; D002692:Chlamydia trachomatis / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D007158:Immunologic Techniques; D008567:Membranes, Artificial; D009344:Neisseria gonorrhoeae / Q000276:immunology; D011136:Polysorbates / Q000494:pharmacology*; D011817:Rabbits
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008567:Membranes, Artificial; D011136:Polysorbates; D002482:Cellulose