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 > > > -- [image: --] David Briggs PhD [image: https://]about.me/david_briggs <https://about.me/david_briggs?promo=email_sig&utm_source=email_sig&utm_medium=email_sig&utm_campaign=external_links>
