ammonium sulfate precipitation & urea - (May/28/2008 )
Hi everybody,
I need to concentrate my recombinant protein after elution from Nichel affinity chromatography in denaturing conditions. Now my protein is in 6M urea plus 250mM imidazole elution buffer. Can I precipitate it with ammonium sulfate? Will my protein aggregate and precipitate even if it's denaturated? Thank you for your help!
I've tried that for protein dissolved in guanidine hydrochloride. I took 1-10 mL of protein solution and mixed it with 100 mL of saturated ammonium sulphate. This way I got 10-100x dilution of guanidine hydrochloride (hopefully protein refolding by rapid dilution?) and simultaneous protein precipitation. It precipitated but protein I was working on was already mess so I'm not sure if it was refolded. 
I hope your protein was correctly refolded because I'm just trying to do the same thing!
I hope your protein was correctly refolded because I'm just trying to do the same thing!
you could refold first then precipitate with ammonium sulfate.
you could also concentrate on an amicon or centricon.
I hope your protein was correctly refolded because I'm just trying to do the same thing!
you could refold first then precipitate with ammonium sulfate.
you could also concentrate on an amicon or centricon.
My protein has a very strong tendency to aggregate. I've tried to concentrate/change buffer with centricon and to refold it by dialysis: in both cases it precipitated. But you were right: it has been not a good idea to refold and concentrate in the same time with ammonium sulfate & dialysis: my protein misteriously disappeared...
Thank you Hey all
I have a very basic question about ammonium sulfate... is there any proof, that salting out with amm.sulf. leave proteins in native state?? I need to measure fluorescence life-times of one protein and I'm afraid, that this salt will cause some demages in it...
I will really appreciate an answer (some suggestions about publications???)!
Thank You in advance!
I have a very basic question about ammonium sulfate... is there any proof, that salting out with amm.sulf. leave proteins in native state?? I need to measure fluorescence life-times of one protein and I'm afraid, that this salt will cause some demages in it...
I will really appreciate an answer (some suggestions about publications???)!
Thank You in advance!
just anecdotal.
i have been using ammonium sulfate to precipitate proteins since i first started working in the lab. after resuspending and dialyzing proteins (enzymes) they all work well.
some protein may denature during the process but that is more an issue of handling than chemistry.
here is a pdf of an article from methods in enzymology, volume 1 (1955):
Also, [attachment=4791:on_colum...efolding.pdf] might be of some assistance:
I have a very basic question about ammonium sulfate... is there any proof, that salting out with amm.sulf. leave proteins in native state?? I need to measure fluorescence life-times of one protein and I'm afraid, that this salt will cause some demages in it...
I will really appreciate an answer (some suggestions about publications???)!
Thank You in advance!
just anecdotal.
i have been using ammonium sulfate to precipitate proteins since i first started working in the lab. after resuspending and dialyzing proteins (enzymes) they all work well.
some protein may denature during the process but that is more an issue of handling than chemistry.
here is a pdf of an article from methods in enzymology, volume 1 (1955):
Is that the first major review on protein solubility-based purification?
i don't know, maybe.
i figured that it would be available in an early volume of methods in enzymology and found it there. there may be something earlier but i didn't look and the references in this review do not appear to be reviews.