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affinity column problems - (Aug/03/2009 )

Hi friends,

I am working with a his tag protein. My problem is my induced product will be stuck in the column for days!! How much ecer I dilute it with the sample application buffer to make it thin it takes days to come down. I don't know what to do??

My SAB composition is,

8M Urea, 1M NaCl, 20% Glycerol, 0.25% Triton X-100, 20mM Imidazole and Tris.

Plz help. Thank you

-scary-

I assume its a Ni NTA column. Use higher concentration of imidazole. Use a gradient up to 250 mM. And why use Urea? It will denature

Best,
TC

scary on Aug 3 2009, 04:51 PM said:

Hi friends,

I am working with a his tag protein. My problem is my induced product will be stuck in the column for days!! How much ecer I dilute it with the sample application buffer to make it thin it takes days to come down. I don't know what to do??

My SAB composition is,

8M Urea, 1M NaCl, 20% Glycerol, 0.25% Triton X-100, 20mM Imidazole and Tris.

Plz help. Thank you

-T C-