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
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-