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<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
