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:[log in to unmask]] On Behalf Of Mark Wilson
Sent: Wednesday, March 29, 2017 1:12 PM
To: [log in to unmask]
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
[log in to unmask] 






On 3/29/17 2:53 PM, "CCP4 bulletin board on behalf of CRAIG A BINGMAN"
<[log in to unmask] on behalf of [log in to unmask]> 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 
><[log in to unmask]> 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:[log in to unmask]] Im  Auftrag von 
>David Briggs
>Gesendet: Mittwoch, 29. März 2017 17:53
>An: [log in to unmask]
>Betreff: Re: [ccp4bb] protein precipitation reg
> 
>It doesn't cost as much as HEPES, iirc.
>On Wed, 29 Mar 2017, 16:36 Keller, Jacob, <[log in to unmask]>
>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:[log in to unmask]] On Behalf Of Roger  
>Rowlett
>Sent: Wednesday, March 29, 2017 11:10 AM
>To: [log in to unmask]
>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: [log in to unmask]
>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>
>
>
>
>
>
>
>
>
>
>
>