Just to point out that whatever buffer you purify your protein into is possibly not the one that will keep your protein happiest. We had the opportunity of testing about 250 proteins in DSF against 26 different buffer / salt combinations (in triplicate, with lots of controls) and found out that about 1/3 of the time the protein was significantly (4C or more) stable in some other buffer system than the one it was in. Ristic, M., Rosa, N., Seabrook, S.A., Newman, J., 2015. Formulation screening by differential scanning fluorimetry: how often does it work? Acta Crystallographica Section F Structural Biology Communications 71, 1359–1364. doi:10.1107/S2053230X15012662
And if we are going to pour scorn and vitriol on Tris, why not mention its large dpKa/dT of 0.03 pH units/deg ? Cheers, Janet -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CRAIG A BINGMAN Sent: Thursday, 30 March 2017 8:54 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg > On Mar 29, 2017, at 4:15 PM, Chun Luo <c...@accelagen.com> wrote: > > In addition to price, the prevalence of Ni purification may be another reason > for Tris popularity. Some His-tagged constructs don't bind to Ni well in > HEPES. I wonder if anyone has similar experience or comments. —Chun No, I have not specifically noted that before. Additionally, why would you use a positively charged buffer on a weak cation exchange resin? The Ni affinity resins, in addition to their noteworthy affinity for various metals, are also weak cation exchange resins. Binding a positively charged buffer to a negatively charged column material can cause pH effects that you probably weren’t expecting. Tris may have some uses. But using it in a mobile phase with Ni affinity columns or as a final sample buffer aren’t the best cases for choosing Tris. I understand it is widely used in aquaculture applications where they are treating tens of thousands of gallons of water, and price of the buffer substance is an actual consideration. Craig
