first of all, you should never make Tris-buffer at pH 11, because that's far outside the buffer range of Tris. For such high pH's you should use either Sodium bicarbonate/Sodium hydroxide or Sodium hydrogen orthophosphate/Sodium hydroxide (see this link: http://delloyd.50megs.com/moreinfo/buffers2.html). Why do you need to have your protein in pH 11 anyway?? This way it will be negatively charged, but you might as well get it into buffer at pH 6 (where it will be positively charged) and then use a cation-exchanger. Is there a specific reason why you want to run a pH-gradient (more complicated) and not a salt-gradient to get your protein out?
Hey,
My suggestion is that pH ranges 6 to 9 should be okay. As Michael pointed, I think pH 11 is way too high. You can check the stability of your protein at pH 11 yourself by running a thermofluor assay.
(Check http://www.ncbi.nlm.nih.gov/pubmed/21472694 for more details)
Best Regards,
Sagar S
Michael-actually I have done salt gradient but it is not giving good purification so I wanted to try pH gradient I think that is more specific..I am trying both Cation as well as anion exchanger...can you please tell me at what pH should my protein elute out ?
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