Simultaneous electroelution of whole SDS-polyacrylamide gels for the direct cellular analysis of complex protein mixtures

Abstract

A novel procedure which allow the rapid screening of complex protein mixtures in cellular assays is described. A device has been developed which allows a convenient, simultaneous electroelution of separated proteins from whole SDS polyacrylamide gels into narrow chambers each containing single or a few protein bands. We have optimized the conditions of the procedure and have obtained an efficient removal of SDS, leading to non-toxic protein fractions in a physiological buffer suited for direct testing in cell cultures. The responses generated by stimulating lymphocytes with the purified products have been compared to the native protein and a corresponding preparation of protein transferred to nitrocellulose particles. The method was used to investigate murine T cell responses to secreted mycobacterial antigens during infection with M. tuberculosis. A immunodominant secreted protein fraction was purified in a semipreparative scale by the procedure and used to immunize mice. The specificity of and lymphokine production by T cells generated in these animals were investigated. The device developed has various applications and provides a tool for the possible identification of new T cell antigens of importance for protective immunity.