protein precipitation / RNA binding factors - (Apr/13/2006 )
Hi, I work with proteins domains that are supposed to bind RNA. All of them have pIs > 9 and are giving me a lot of problems in trying to purify them. They are mostly insoluble, but I think that the main problem is precipitation when I try to concentrate ( some related domains with acidic or neutral pIs don't give me any kind of problem)
try using a buffer with pH 2 units higher than the pI. that would mean a buffer of ph 11 !! maybe this is too high if you are wanting to do functional studies, but worth a try.
Try dialyzing the protein into a buffer like: 25mM Tris-Cl, with about 100 mM NaCl and 50% glycerol. The glycerol should help with freezing. Remember, however, that your protein volume will concentrate by half, so dilute if neeeded accordingly!
Then, another question I have is, given I could concentarte my protein in this buffer with such a high amount of glicerol, do I have any possibility of removing the glicerol without precipitating then the protein? Unfortunately, I cannot have my protein with so much glycerol in the fina buffer because it is a great containant in my further studies...