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<mailto: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<mailto: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<mailto: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<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Roger Rowlett Sent: Wednesday, March 29, 2017 11:10 AM To: CCP4BB@JISCMAIL.AC.UK<mailto: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<mailto: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>
