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protein precipitation after IMAC - protein purification (Mar/01/2005 )

I'm working on 35kDa protein with HIs-tag N-terminal. After purification on Ni-column (Qiagen) I usually set up the dialysis o/n and the protein always precipitates. It doesn't matter if I use Tris, HEPES, CAPS. I tried pH 4-10. I add 20% glycerol, Bme, I try to elute using low pH instead of imidazole, I increased the NaCl concetration, also dilution of my eluted protein, I did the dialysis at 4C and also at room temp... nothing helps. Only what I can say - yes I;m sure I have my protein, it's really pure on the SDS gel and I get quite a lot of it after IMAC. Do you have any ideas what else I could try?? Thanks in advance for your help!!! Agata


His tag someimes affects the folding of the proteins. Try deanturing the sample with urea or guanidine.HCL and thereafter renaturing it.

It appears that passage thorugh N- sepharose is affecting the affecting. See if you can employ any other method of purification instead.