visualised by sds page followed by western blotting using anti histidine antibody as primary antibody and rabbit anti mouse ipg hrp conjugate as secondary antibody the recombination groel protein is purified by ni nta chromatogramphy under denaturing conditions briefly the protein sample is added to the charged gel and allowed to bind to the gel for 30 min at room temperature with gently agitation the gel is pelleted by centrifugation at 500 g for 2 minutes and the supernatant is carefully decanted the gel is washed with wash buffer 3 times and the protein is finally eluted with elution buffer using 0 5 m imidaole after purification the protein is refolded in refolding buffer refolding is initiated by rapid dilution 10 times of the purified protein 100 u03bcg final concentrations in refolding buffer 50 mm tris 1 mm edta 1m arginine 1 mm gsh 0 8 mm gssg ph 7 5 solution is kept stirring during dilution and is then incubated for 24 h at 4 u00b0 c without stirring the refolded preparation is dialyzed extensively to eliminate arginine and urea against dialysis buffer for 24 h with buffer changes every 12 h the dialyzed sample is centrifuged and concentrated by amicon filtration the immune response of recombinant groel is evaluated by injecting 50 u03bcg protein mouse intraperitonially on 0 7 th and 28 th day seven days after the last injection the mice are sacrificed and serum is collected for antibody titre antibody titre is determined by elisa using 96 well microtitre plates previously coated with 1 u03bcg of dnak protein well and blocked with bsa different dilutions.