In article <3v3k25$t8u at sifon.cc.mcgill.ca>, anne-claude
<gingras at medcor.mcgill.ca> wrote:
>> Hi,
>> I am having a tough time trying to purify his-tagged proteins using
> QUIAGEN system. First of all, the expression levels I am getting are
> quite low (<200=B5g/L), but still, the protein is >90% in inclusion
> bodies. When we purify this protein under denaturing conditions, we see
> contaminant bands at an higher molecular weight. Finally, almost all
> the recovered protein stick to dialysis tubing and cannot be recovered
> at the final stage.
>> Does somebody have nice tricks to try in order to get a protein in the
> soluble fraction ? Did somebody ever noticed the presence of other
> bands on a Coomassie gel ? What can I do to increase the yield ? Any
> idea of how I can do to avoid the protein to stick to the tubing ?
>> I will be most grateful if somebody can answer to one of more of my
> problems. Thank you a lot !
>> Anne-Claude (gingras at medcor.mcgill.ca)
Hi,
for avoiding contaminations try first the isolation of inclusionbodies,
this
makes you get rid of alot of contaminations. Additional you can add 10-20
mM
mercaptoethanol to all the chromatography-buffers, avoiding contaminants
that are bound with disulphide bonds.
To avoid sticking to the tubing:
Don«t use dialysis-tubing made of cellulose hydrophobic proteins will stick
to it and you will loose a lot. There are tubings of cellulose-acetate
which overcome
this problem. I usally get yealds greater 90% even with very hydrophobic
proteins.
Second you may add some tensides, like CHAPS, octylglucoside (both mild and
non denaturing)
I get usually greater 95% active protein on dialysis from 6M GuCl with
octylglucoside.
Hope this helps,
Stephan
Stephan Witte
Inst. of Immunology
University of Constance
stephan.witte at uni-konstanz.de
