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affinity column problems


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#1 scary

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Posted 03 August 2009 - 02:21 AM

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

#2 T C

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Posted 03 August 2009 - 02:36 AM

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

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TC

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






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