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
-----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark Wilson Sent: Wednesday, March 29, 2017 1:12 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg I heartily concur with Craig. Tris can be a dangerous buffer for many reasons, including those listed below. In addition, as a primary amine, it can complicate work with metalloproteins and has moderate nucleophilicity. There is almost always a better buffer choice than Tris. Best regards, Mark Mark A. Wilson Associate Professor Department of Biochemistry/Redox Biology Center University of Nebraska N118 Beadle Center 1901 Vine Street Lincoln, NE 68588 (402) 472-3626 mwilso...@unl.edu On 3/29/17 2:53 PM, "CCP4 bulletin board on behalf of CRAIG A BINGMAN" <CCP4BB@JISCMAIL.AC.UK on behalf of cabing...@wisc.edu> wrote: > > > >There are almost always better choices than Tris buffer. > > >Mo Cleland used to call it “Trash” buffer. He is no longer with us, >but today I will happily carry that flag in his honor. > > >Tris may show up in your crystal structure, especially at carbohydrate >binding sites. >Tris may be a surprisingly strong competitive inhibitor in your enzyme >assays, especially as above. >Tris has an absolutely miserably bad change in pKa vs. temperature. It >is larger than -0.03 pKa/dT(C). It can be a catastrophically bad >choice for flash-freezing protein aliquots. > > >If you taken the time and incurred the expense of preparing a >macromolecular sample for crystallization studies, and you are worried >about the price difference between Tris and HEPES, in my opinion you >are absolutely worried about the wrong things. > > >Why are people substantially concerned about the buffering capacity of >a buffer for final sample preparation? You have a purified protein, >presumably without substrate present. Exactly what do you think is >generating or absorbing hydrogen ions in that solution? Oxidation of >reducing agent should be about the only thing that is taxing the >buffer. From the example below, oxidation of 5 mM BME will put some >pressure on the buffer, but unfortunately Tris accelerates the >oxidation of BME relative, to, say, HEPES. And surely you aren’t just >letting the protein sit and oxidize in the refrigerator? Oh you might >be since when you tried to snap freeze it in Tris, it turned into >cooked egg white because the pH went to over 10 before it vitrified. >(http://www.sciencedirect.com/science/article/pii/S0031942200801429) > > >Isn’t the whole point to use a small amount of buffer so you can easily >push the pH around in crystallization screens? (At which point the >sample is usually in 100+ mM buffer.) > > >On Mar 29, 2017, at 2:03 PM, Hughes, Jon ><jon.hug...@bot3.bio.uni-giessen.de> wrote: > >...it's just a wonderful tradition! there's an interesting description >of the history of tris in maniatis.... >cheers >jon > >Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von >David Briggs >Gesendet: Mittwoch, 29. März 2017 17:53 >An: CCP4BB@JISCMAIL.AC.UK >Betreff: Re: [ccp4bb] protein precipitation reg > >It doesn't cost as much as HEPES, iirc. >On Wed, 29 Mar 2017, 16:36 Keller, Jacob, <kell...@janelia.hhmi.org> >wrote: > > >A bit off topic, but I’ve always wondered how TRIS got so popular what >with it’s pKa of 8.3—does anyone know? > >JPK > >From: CCP4 > bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger >Rowlett >Sent: Wednesday, March 29, 2017 11:10 AM >To: CCP4BB@JISCMAIL.AC.UK >Subject: Re: [ccp4bb] protein precipitation reg > > > > > >What are you dialyzing against? Your storage solution should typically >be buffered away from the pI and contain at least a small amount of >kosmotropic salt, e.g. NaCl. Some proteins will require additional >stabilizing/solubilizing agents such as glycerol or reducing agents. >FYI, Tris-Cl, pH 7.5 has very little buffer capacity (about 15% of the >total concentration in the acid direction). We typically use Tris-Cl pH >8.0, which is closer to the Tris pKa and has good buffer capacity for >both acid and base. For pH 7.5 we would typically use HEPES as the >storage buffer. > >_______________________________________ >Roger S. Rowlett >Gordon & Dorothy Kline Professor >Department of Chemistry >Colgate University >13 Oak Drive >Hamilton, NY 13346 > >tel: (315)-228-7245 >ofc: (315)-228-7395 >fax: (315)-228-7935 >email: rrowl...@colgate.edu >On 3/29/2017 9:38 AM, Akilandeswari Gopalan wrote: > > >Dear all, >I am a PhD student doing structural studies on a few proteins from >Mycobacterium tuberculosis. The gene encoding the proteins I work on >are cloned into pet22b with c terminal His tag. the proteins are >expressing well. upon purification I am getting good yield of protein >but during dialysis, the proteins precipitate. Kindly suggest some >solutions to avoid aggregation. pI of one protein is 9.7 and that of >the other is 5.6 > >I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM >beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer >with 20-30mM imidazole for washing and 300mM imidazole for eluting the >proteins. > > > >Thank you > >Regards > >Akila > > > >-- >Akilandeswari G > > > > > > > > > > > >-- > > >Fehler! Es wurde kein Dateiname angegeben. ><image001.jpg> > > >David Briggs PhD > >Fehler! Es wurde kein Dateiname angegeben. ><http://angegeben.about.me/david_briggs>about.me/david_briggs ><http://angegeben.about.me/david_briggs> > ><image002.jpg> > > > > > > > > > > >
