In article <SS32-1505971739260001 at mac65.msb.le.ac.uk>, SS32 at le.ac.uk (MacFS User) says:
>>hello
>i'm trying to purify a HIS TAGGED protein on nickel-NTA RESIN, from
>qiagen. i'm having very little binding and very poor recovery . i need to
>purify under native conditions , does anyone have any suggestions please.
>please reply to ss32 at le.ac.uk>thanks
>shital
>leicester university
You neglected to say whether or not the His tag was on the N or C terminal
of your protein. This can make a difference as to the accessibility of
the tag to the environment. Unfortunately, sometimes the tag is buried in
the protein and the nickel resin does not interact. Did you check to
see if the protein is flowing through when you attempt to load the column?
Tom
