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Dear All

I am working with HIS – tag protein in N-terminal (hexa histidine). The protein from Prokaryotic origin cloned into pET30a+ vector and expressed in E.coli BL21 cells. The expression was good. I am trying to purify a protein using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same as buffer A but 250mM imidazole). I eluted the protein in step wise gradiant. the protein was less binded in Ni affinity IMAC column because eluted fraction contain less amount and the protein remain present in the Flow through. Can any one suggest how to increase the binding affinity of the protein and how to purify the protein. The protein PI was 6.33