I personally like TRIS for the first few steps of purification and then change to something else during my last dialysis step. I mostly work with bacteria and they often produce lysates that have pH's that are too acidic for good nickel affinity chromatography, which is why I use 100 mM TRIS pH > 7.8 at this point (if I remember correctly, histidines won't be properly charged and won't bind well to the nickel ions at pH < 7.6). 10 or 50 mM of most buffers might not actually buffer a fairly concentrated bacterial lysate and therefore produce a solution that is more acidic than expected.
I also run size exclusion chromatography in 100 mM TRIS to reduce the cost as 1L of 100 mM HEPES is fairly expensive (pH can be lowered at this point depending on the protein's PI, but I like to have the solution strongly buffered). After SEC I will use 10 mM HEPES (or other appropriate buffers) for the final dialysis step. This reduces the cost and works well for me. BTW, TRIS is obviously not a good buffer choice for ion exchange chromatography (which relies on precise pH differences between two buffers) because the pH of a TRIS solution will change with even small fluctuations in temperature. Best regards, Tony ------------------------------------------------------ Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0AAAAJ&hl=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5> ________________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chun Luo [c...@accelagen.com] Sent: 29 March 2017 22:15 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg 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
