PURIFICATION OFMURINE HEAT SHOCK PROTEIN90-BETA (84 KDA) FROMMICE SPLEEN

Murine heat shock protein 90 beta isotype (84kDa) was purified from the cytoplasm of BALB/c mice spleen cells. Since this protein dose not have an in vitro activity assay and the presence of protein with molecular weight equal or near 84 kDa was followed in each purification step. Cytoplasm fractions of spleen cells contained proteins of molecular weight (MW) ranged from 86.0 to 20.9kDa as determined with SDS-PAGE analysis. The ammonium sulfate 70% saturation precipitate of cytoplasm fraction exhibited protein bands with molecular weight ranged from 40.7 to 86.1kDa. Ion exchange chromatography with DEAE-Cellulose column for the ammonium sulfate precipitated cytoplasm proteins produced one prominent peak, eluted between 0.5 and 0.7M NaCl. This peak contain four proteins with MW ranged from 19 to 83.1kDa. During gel filtration through Sepharose 6B column for the peak eluted from DEAE-cellulose column, a peak with estimated molecular weight of 87kDa was selected among others produced. Second round of gel filtration through Sephacryl S- 200 was conducted for the 87kDa selected fractions eluted from the previous gel filtration step. The presence of one peak was emphasized and its estimated MW was 83.176 kDa. The molecular weight and purity of this protein was checked again with SDS-PAGE 10%. The identity of the purified proteins as Hsp90β was confirmed with immunofixation assay by gel electrophoresis on 1%agarose using standard.