Hello everyone.
My protein has the pI of 6.1, MW of ~25kDa and very hydrophilic property. I confirmed the solubility(>90% of soluble) using various expression tests. I expressed recombinant protein(C-terminal 6X His-tagged) in BL21(DE3) and purified first using Ni-NTA column(starting buffer : 20mM Tris-HCl pH 7.0, 0.2M NaCl, 10mM imidazole). Because of some impurities, I decided using Q column and started desalting and buffer exchange with PD-10 column(buffer : 20mM Tris-HCl pH 8.0, 1mM DTT, 0.1mM EDTA). At that point, I observed immediately some hazy in eluted sample. I added final 0.2M NaCl in sample and also observed clarity(and then remove the aggregate by centrifugation). Although I finally made an attempt gel filtration using superdex 75(buffer 20mM Tris-HCl pH 8.5, 0.2M NaCl), I only observed aggregate. My protein has not thrombin cleavage site and not sequential hydrophobic patch. I anticipate some advice. Thanks
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