A simple method of electroelution of individual protein bands from SDS polyacrylamide gels for direct study in cellular assays.
A very simple and effective procedure which allows simultaneous electroelution of separated proteins from SDS polyacrylamide gel into small quantity of elution buffer is described. Elution parameters have been optimized for maximum possible recovery (50-60%). Protein fractions were collected in physiological buffer and an efficient removal of SDS have been obtained, thus fractions collected were suited for direct testing in cell cultures. Method was used to investigate human T-cell responses to purified secreted M tuberculosis H37Rv proteins. Eight low molecular weight (M.w. range 10 kD to 25 kD) culture filtrate proteins were purified in quantities, sufficient for immunological characterization. Lymphocyte proliferative responses and cytokine release pattern from tuberculosis patients, healthy contacts and healthy controls were studied on stimulation with purified culture filtrate proteins. Immunologically important M.tuberculosis proteins were identified by using this method. This approach should be applicable to the rapid identification and characterization of any interesting T cell antigen.
Bhaskar
S
S
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India. sangeeta@nii.res.in
Dutt
S
S
Mukherjee
R
R
D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification*; D016207:Cytokines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008213:Lymphocyte Activation; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D014397:Tuberculosis, Pulmonary / Q000276:immunology*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016207:Cytokines