his tagged protein

Koen A.L. De Smet k.desmet at ic.ac.uk
Mon May 19 08:53:59 EST 1997

Tom McCloud wrote:
> MacFS User wrote:
> > > 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.
> >
> A colleague here does what you have asked about, so I am passing along
> his suggestions:
> 1) check the pellet to see if you are really solubilizing the protein.
> Try conditions like 50mm, 250mm, 1M NaCl,  with/without detergent,
> with/without betamercaptoethanol (do NOT use DTT - it binds strongly to
> the NI column)
> 2)do not use DTT or EDTA if can avoid it.  If you must use it to
> resolubilize, then wash by diluting out, and spin several times before
> loading onto the column.
> 3)try phosphate buffer or lower strength TRIS or HEPES for loading. In
> some cases amines, including the buffers, can interact with the column
> and prevent binding.
> 4)If you can't get the protein off the column, supplement with
> imadazole, and try additional NaCl or detergent or glycerol to get the
> bound protein to elute.
> 5)Try batch elution: empty the colum into a tube that can be gently
> rocked, filter off the resin in the morning, measure protein in the
> solution.
> 6)Presence of DNA or macromolecules may inhibit proper binding.  Some
> people treat with a small amount of DNase to chop up DNA into small
> pieces.  High viscosity is a problem.  Large particulates, aggregates of
> proteins should be spun out before loading.
> 7)If your protein is simply not soluble, then try putting your tag on
> the opposite end, i.e. switch N term for C-term tag. Sometimes the end
> which the tag is on makes a big difference in solubility.
> 8)As a last resort, if the protein simply will not elute from the resin,
> strip the column with EDTA.  This strips the NI plus the protein, but
> you should be able to measure the eluate for protein content---it just
> has a lot of NI in it.   And the column can be regenerated.
> 9)The protein structure can be 'loosened - up' in 1 to 2 M urea or
> guanidine, which really does not denature.  Maybe even load the column
> in this.    But when proteins have been totally denatured then
> everything tends to bind to the NI column.
> Good luck.  This info is second hand, but I have tried to accurately
> pass my colleagues thoughts along.     Tom McCloud  SAIC/Frederick
> Cancer Research

May I add to other factors that helped in my case:
1/ ensure that the crude extract or binding buffer has a pH of 8.0 or 
2/ add the Ni-resin to the extract and shake gently for an hour to leave 
plenty of time for binding. Then put the whle slury in a column and wash 
and elute.

And of course, collect all fractions and compare with a crude extract to 
find out if binding occured.



        Dr Koen A.L. De Smet
        Research Fellow
        Department of Medical Microbiology
        Imperial College Medical School at St Mary's
        Norfolk Place
        London W2 1PG
        Great Britain
        Tel: (+44)-(0)171-594 3946  
        Fax: (+44)-(0)171-262-6299   
        Email: k.desmet at sm.ic.ac.uk

"If we knew what it was we were doing,
 it would not be called research, would it?".   --   A.Einstein

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