Simultaneous electroelution of whole SDS-polyacrylamide gels for the direct cellular analysis of complex protein mixtures.
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.
Andersen
P
P
Statens Seruminstitut, Bacterial Vaccine Department, Copenhagen, Denmark.
Heron
I
I
D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D007114:Immunization; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D010047:Ovalbumin / Q000276:immunology; D012967:Sodium Dodecyl Sulfate; D013601:T-Lymphocytes / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D012967:Sodium Dodecyl Sulfate; D010047:Ovalbumin