How to keep recombinant protein stable and soluble?
bassuk at u.washington.edu
Sun Nov 17 18:00:37 EST 2002
On 12 Nov 2002 dustjohn at hotmail.com wrote:
> Can somebody please comment on the following:
> After capturing a his-tagged protein on Ni-NTA, it is eluted with 250mM imidazole, 500mM NaCl and 50mM sodium phosphate at pH=8.0. The protein in this sample appears to be soluble (although I've never left it for extended periods of time) but after 3 rounds of concentrating via centrifugation in a centricon device and buffer exchange (to a low-salt buffer for anion exchange), there are large aggregates of protein that have formed. After addition of 20mM 2-mercaptoethanol and incubation on ice for 10 minutes, much of the aggregated protein disappears. Then subsequent anion exchange yields his-tagged protein that elutes at 250mM NaCl; this sample once again aggregates if left at 4 celsius overnight. Are there any secrets to keeping such a protein soluble? Should we replenish reducing reagent continually? Should we add glyercol or switch to a slower dialysis period?
More info is needed:
* What is your recombinant system?
* What is the buffer that your sample is prepared in?
* From what concentration are you starting at before centriplus-ing?
* What is the pI?
* How many cysteines?
More information about the Proteins