Hello everyone, I'm trying to purify his-tagged protein under denaturing conditions using Guanidine Hydrochloride (Protocol from GE Healthcare-attached below).
Though I'm getting my protein in elutions the concentration is very less, since the protocol doesn't say about binding conditions with the column (RT or 4C) I have proceeded to bind at cold temperature. Will it help me if I'm doing all these processes at RT?
Can you please let me know how can I go about binding in this case from your experience!
Thanks in advance.
normally these purifications are carried out at 4°C, have you tried to calculate a purification yield? Did you had protein in your Flow through or Wash fractions?
Dear Venkat
you can certanilly try to perform the purification at RT because in denaturing condition you do not have protease activity and therefore is less important be at 4°C, however i do not think that the temperature will change a lot the binding ability of your protein.
Just some questions:
Did you check the final pH of your buffers? are 8?
Is your protein rich of cysteines or really hydrofobic (eg membrane protein?)
because protein aggregation may reduce the protein binding ability on the resin.
Did you checkd by SEC the aggregation state of the eluted protein?
ciao
Manuele
Dear Venkat,
GuHCl will work under RT and cold conditions. It is preferred to have IB purified for refolding due to contamination from proteases after elution from the column.
The questions you have to ask have been answered before. I would do basic homework, rethink it and post the question again. People will help you if you present a specific problem instead of a general question which can give you contradictory answers.
Wes
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