Exactly. Tris is very cheap. HEPES not so much. On the other hand,
zwitterionic buffers have significant advantages in terms of controlling
inorganic anion or cation concentrations.
_______________________________________
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 11:53 AM, David Briggs wrote:
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
--
--
David Briggs PhD
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>
