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