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
above.
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.
Koen
_______________________________________________________________
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.ukhttp://www.sm.ic.ac.uk/medmicro/home.htm
_______________________________________________________________
"If we knew what it was we were doing,
it would not be called research, would it?". -- A.Einstein
_______________________________________________________________
