Help with Protein Precipitation
mw132 at mole.bio.cam.ac.uk
Sun Jun 24 08:48:49 EST 2001
in my case I solved a similar problem with a series of very small
scale dialysis experiments to see what I needed in my buffer to keep my
periplasmic protein soluble. I tried a range of NaCl concentrations,
pH, and so on and so on, which didn't help at all. After a lot of
experiments I found that a very small amount of detergent made the protein
stable in 10mM Tris pH8. 0.01% Triton X100, Tween 20 or NP40 were good
but I finally selected 0.01% octyl glucoside because it is thought to be
best for MS. But the microdialysis experiments were what made finding out
possible. Regards, Mike.
> Hello all!
> I'm currently purifying a 6xHis tagged recombinant protein from
> E. coli extracts using Ni-NTA agarose. The purifications are going fine
> (thankfully), but when it is time to dialyze out the 6M urea that the
> protein samples are in, the protein clumps up in the dialysis bag.
> Following precipitation, it is a constant headache of resuspending the
> ppte in 6M urea, hence increasing the volume, re-dialyzing and I'm
> getting very low protein yields in LARGE volumes of buffer. I have
> tried a gradient dialysis going from 6M/10mM Tris -> 5-4-3-2-1-0 M
> urea/10mM Tris., and it still precipitates. This is a membrane protein,
> so naturally it prefers a lipid environment - but someone else must be
> experiencing such problems (and working with membrane proteins) and may have
> solution to this problem?
> Do you think the addition of some NaCl would minimize precipitation upon
> dialysis? (someone suggested it, and I tried it and it seemed to make
> matters worse).
> Thanks in advance for a response!
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