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6 His tagged protein purification - (Nov/28/2005 )


I have a problem in purification of 6 His-tagged protein (17kD)


The recombinant protein is expressed in low level.
I changed induction condition alot in ' temerature, IPTG concentration' .

However i couldn't obtain more protein.
So i gave up to get more. then neway i went through purification step.

i eluted the 6His tagged recombinant protein then i ran it on 15% SDS PAGE.

but there were several MAJOR bands.

Why is it happening? Isnt 6 his tagging system specific?
I dun know how to approach to this problem.

help~ some experts on His-tagged protein prufication~!!!!! smile.gif



e coli does contain some proteins that may occasionally elute from the column, but I would check a couple other things first:

have you done anything to change your buffers and such? perhaps your POI is not as soluble as you think it is...have you run any other fractions to see the difference?

how do you know the protein is expressed at a low level?

what are the sizes of the major bands? could they be oligomers?

are you sure you are getting the protein at all? have you sequenced the clone to be positive everything is in frame? have you checked with a western or something for your protein to be quite sure the right one is even there?

if you can answer some of these questions perhaps we can help you pinpoint the problem

OH, hey, I made this mistake once: if it is driven by a T7 promoter, you won't get much of anything if you are growing it up in the wrong strain...what is your vector and your e coli strain?