A simple method of electroelution of individual protein bands from SDS polyacrylamide gels for direct study in cellular assays

Citation
S. Bhaskar et al., A simple method of electroelution of individual protein bands from SDS polyacrylamide gels for direct study in cellular assays, J IMMUNOASS, 21(4), 2000, pp. 355-375
Citations number
20
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Immunology
Journal title
JOURNAL OF IMMUNOASSAY
ISSN journal
0197-1522 → ACNP
Volume
21
Issue
4
Year of publication
2000
Pages
355 - 375
Database
ISI
SICI code
0197-1522(2000)21:4<355:ASMOEO>2.0.ZU;2-2
Abstract
A very simple and effective procedure which allows simultaneous electroelut ion of separated proteins from SDS polyacrylamide gel into small quantity o f elution buffer is described. Elution parameters have been optimized for m aximum possible recovery (50-60%). Protein fractions were collected in phys iological buffer and an efficient removal of SDS have been obtained, thus f ractions collected were suited for direct testing in cell cultures. Method was used to investigate human T-cell responses to purified secreted M.tuber culosis H37Rv proteins. Eight low molecular weight (M.w. range 10 kD to 25 kD) culture filtrate proteins were purified in quantities, sufficient for i mmunological characterization. Lymphocyte proliferative responses and cytok ine release pattern from tuberculosis patients, healthy contacts and health y controls were studied on stimulation with purified culture filtrate prote ins. Immunologically important M.tuberculosis proteins were identified by u sing this method. This approach should be applicable to the rapid identific ation and characterization of any interesting T cell antigen.