Hi,

I am trying to purify a kinase protein (theoretical pI = 9.5) by HIS-select Ni-affinity column, washing with 50mM Tris HCl (pH 7.5), 0.5M NaCl, 20mM imidazole (buffer A) and a gradient elution with the same buffer + 500mM imidazole (buffer B). The protein becomes precipitated within 10 minutes after the elution, especially, in the elution tubes containing more than 0.5mg/ml concentration. I tried adding 5% glycerol in the elution buffer, but it did not work. Adding 10% glycerol showed degradation of protein in the SDS-PAGE.

When I centrifuged the sample tubes having precipitation, collected supernatant, mixed with other elution tubes containing soluble protein, and then concentrated the total volume (around 18ml) to 5ml ( for gel filtration), there was no precipitation. However, after gel filtration (buffer: 20mM Tris, 300mM Nacl, pH 7.5), the protein yield was very less as compared to the loaded sample. The protein's molecular wight is 42kDa, and i used S200 column for gel filtration.

Any thoughts??