Hi,
There are proteins which, even with a His tag, elute at 40-60 mM
imidazole. Ideally, you should try to use 'real' chromatography (i.e.
FPLC) to monitor the progress of your elution. For weakly binding
proteins, you would lyse the cells in say, 50 mM phosphate/300 mM NaCl
with protease inhibitors, load the stuff on the column and elute with
0-300 mM gradient imidazole. There should be two peaks, one at 0-30 mM
containing all sorts of crud and one higher than that, with (mostly)
your target protein in it. Please note that IMAC is NOT the most
rigorous way to purify proteins, it is a good first step but it often
results in ~60-70 % pure protein which needs lots of further work-up.
Importantly, TRIS is NOT recommended for IMAC (in fact most amines would
interfere, including commonly used ammonium sulphate) - phosphate is
MUCH more advised. Make sure you have no more DTT than 0.5-1 mM (and it
is better to have imidazole).
As silly as it sounds, make sure that your protease inhibitor cocktail
is EDTA-free.
His antibody is OK to use for general detection of the tag, however for
the most reliable test your best bet is mass-spec. His6-antibody would
react with even the smallest fraction of the intact protein in a sample
that may mostly contain untagged stuff and will give you a false
positive.
Good luck,
Artem
"J. Arturo García-Horsman" wrote:
>> John wrote:
>> > I have expressed my gene of interest (0.8kb) in a C-terminal His-tagged
> > expression vector. I used 200mM Tris SO4 pH7.9 in resuspending the cell, and
> > obtained the cell free extract by sonication.
> >
> > Then I would like to purify the protein by using Qiagen Ni-NTA spin Kit (Cat
> > No. 31314). However I found that the eluate are the same as the portion
> > saved from the washing step. That is all the bands came out instead of a
> > single band.
> >
> > I have tried to resuspend the cell in the lysis buffer (as described in the
> > protocol), however the result was the same.
> >
> > The expression of the protein of my interest has been confirmed by
> > Activity-stained PAGE.
> >
> > So, would anyone give me some suggestions? Thank you very much.
> >
> > Johnny
> >
> > ---
>> Hello Johnny,
>> 1) Is your protein still intact? Has the histidine tag remained at C-terminous?
> You can check that, if you haven't, by western with anti-6Xhis antibody. It may
> be that your protein is loosing the tag post-expression or during lysis
>> 2) Are you loading your sample in low imidazol? something like 10-20 mM imidazol
> in the loading buffer usually helps in avoiding unspecific binding.
>> 3) How many times you washed your column? you can wash it more before elution to
> eliminate all the unspecific binding. Is your protein present in all fractions?
> (i.e. washes, eluate). Is it coming out at all from the column?
>> 4) I guess that your avoiding all the components that could compete
> for/inactivate the comunn.
>> 5) . . .
>> Cheers,
>> Arturo.
> _______________________________________________________________
>> Arturo Garcia-Horsman, Ph.D.
> Research Specialist
> Department of Pharmacology and Toxicology
> University of Kuopio
> Canthia, Harjulantie 1 A, 1. krs.
> 70210 Kuopio, Finland
> Tel: +358-017-162422
> Fax: +358-017-162424
> E-mail: Arturo.Garcia-Horsman at uku.fi
--
|Dr. Artem Evdokimov Protein Engineering |
| NCI-Frederick Tel. (301)846-5401 |
| FAX (301)846-7148 |
|eudokima at mail.ncifcrf.gov |
|http://www.ncifcrf.gov/plague |
