how to elute "only" my recombinant protein - final elutes comes with non-specific proteins... (Jun/04/2011 )

Hi,

How can I remove additional proteins of about 90-100 kd in the final elute. My protein size is about 46 kd and I use His based protein purification. (Qiagen)

Data:

Expression is good but for solubility I induced the culture with 0.1mM IPTG and grow further at 16 degree Celsius

Lysis buffer composition is tris based- 50mM tris, 500mM NaCl, 15mM BME,10% glycerol, lysozyme + PMSF having overall pH 6.4 (coz my protein does not bind with column at higher pH)

Washing with 80mM imidazole
And elution with 250 mM Imidazole

In washing step, non-specific proteins which I mentioned earlier are washed slowly but after using 170 ml wash buffer my protein unbinds with the column and comes slowly-slowly with non-specific proteins….

And this is my problem… how to elute only my recombinant protein.

Kindly help me,

I am not sure but I would think about
maybe pooling the fractions, concentrating it and then running it through a gel filtration (size exclusion) column such as S-200.

I don't think it is common to have pure protein from the 1st column, at least that is my experience.

May I also recommend wiki:

http://en.wikipedia.org/wiki/Polyhistidine-tag

Wash buffer better has lower imidazole concentration ( e.g., 10 or 20 mM ). The elution better be done with a gradient from 20 mM to 300 mM of imidazole. That'll give you a better elution of your protein and better separation from non-specific proteins.

Also,many times Ni-NTA purification alone won't give you pure protein. In that case, SEC (gel filtration)can help if your protein size is significantly different from non-specific proteins like in your case.

If just one non-specific protein like in your case, maybe a centrifugal filter with 50KD cut-off can help you get rid of the non-specific protein.

thnx...for ur consideration
ys indeed both method r good for me...

but can u all tell me... is there any way ,,i mean not using SEC or cutoff filter,,,,