Please have a look at the very elaborated/detailed discussion by Martin
Chaplin on chaotropes and kosmotropes
http://www1.lsbu.ac.uk/water/kosmotropes_chaotropes.html
and on the Hofmeister series
http://www1.lsbu.ac.uk/water/hofmeister_series.html
and *note the diference* in the way the ions
On 03/30/2017 08:10 AM, mesters wrote:
If the pI of the protein is below the pH of the buffer (net negatively charged
protein), optimum stabilization (salting out; lower solubility) of the
macromolecule is achieved by combining a kosmotropic anion with a chaotropic
cation, e.g. Ammoniumsulfate
n
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> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Akilandeswari
> Gopalan [akilaibt2...@gmail.com]
> Sent: 30
Hi Akila,
I'm curious about your choice of pET22b. If you cut out the signal peptide
that comes with the vector and express your protein in the cytoplasm, then
what I am about to say doesn't apply. However, if you are exporting the
protein into the periplasm, have you considered doing osmotic shock
If the pI of the protein is below the pH of the buffer (net negatively
charged protein), optimum stabilization (salting out; lower solubility)
of the macromolecule is achieved by combining a kosmotropic anion with a
chaotropic cation, e.g. Ammoniumsulfate (most successful salt)!
/For your pI 9
L.AC.UK] on behalf of Akilandeswari
Gopalan [akilaibt2...@gmail.com]
Sent: 30 March 2017 07:02
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein precipitation reg
Dear all,
I have used the following buffers for purification and dialysis. this is fyi.
Lysis buffer:
25mM Tris pH 7
B@JISCMAIL.AC.UK] on behalf of Chun Luo
[c...@accelagen.com]
Sent: 29 March 2017 22:15
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg
In addition to price, the prevalence of Ni purification may be another reason
for Tris popularity. Some His-tagged construc
Darn it, you're right.
JPK
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes,
Jon
Sent: Thursday, March 30, 2017 4:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg
yes, "oil of v
Apple products
-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark
Wilson
Gesendet: Mittwoch, 29. März 2017 22:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg
I heartily concur with Craig. Tris can
IL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg
Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an
old name for sulfuric acid.
> On Mar 29, 2017, at 8:40 PM, Keller, Jacob wrote:
>
>> And if we are going to pour scorn and vitriol on Tri
tural Biology
Roosevelt Drive
Oxford
OX3 7BN
Original message
>Date: Thu, 30 Mar 2017 11:32:05 +0530
>From: CCP4 bulletin board (on behalf of Akilandeswari
>Gopalan )
>Subject: Re: [ccp4bb] protein precipitation reg
>To: CCP4BB@JISCMAIL.AC.UK
>
> Dear all,
>
Dear all,
I have used the following buffers for purification and dialysis. this is
fyi.
*Lysis buffer*:
25mM Tris pH 7 or 7.5 or 8
100-500mM NaCl (increase in salt concentration increased precipitation of
the protein in the column itself)
*5mM Beta mercaptoethanol *
*0.5% Triton X 100 *
I ha
Hi Akila,
In addition to what others have asked about the dialysis buffer, a few
more comments that might help to decide next steps because the
precipitation (note precipitation and aggregation are related but not
synonymous) may be due to several different or related reasons:
1) At what stage ar
Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an
old name for sulfuric acid.
> On Mar 29, 2017, at 8:40 PM, Keller, Jacob wrote:
>
>> And if we are going to pour scorn and vitriol on Tris, why not mention its
>> large dpKa/dT of 0.03 pH units/deg ?
>
> Hah! That
>And if we are going to pour scorn and vitriol on Tris, why not mention its
>large dpKa/dT of 0.03 pH units/deg ?
Hah! That's what many people are doing when they make buffers: pouring vitriol
(HCl) on TRIS! I prefer to pour concentrated HEPES, and get two buffers without
adding any extra Cl-.
Behalf Of CRAIG A
BINGMAN
Sent: Thursday, 30 March 2017 8:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg
> On Mar 29, 2017, at 4:15 PM, Chun Luo wrote:
>
> In addition to price, the prevalence of Ni purification may be another reason
&
> On Mar 29, 2017, at 4:15 PM, Chun Luo wrote:
>
> 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
No, I have not spec
CP4BB@JISCMAIL.AC.UK] On Behalf Of Mark
Wilson
Sent: Wednesday, March 29, 2017 1:12 PM
To: CCP4BB@JISCMAIL.AC.UK
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 pr
27;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
>Betreff: Re: [ccp4bb] protein p
SCMAIL.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,
mailto:kell...@jane
: 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
nt:* 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
sm
CP4BB@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
>
) 706-7457
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller,
Jacob
Sent: Wednesday, March 29, 2017 11:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein precipitation reg
A bit off topic, but I’ve always wondered how TRIS got so popular what with
it’s pKa
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
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
And what are you dialyzing it against?
JPK
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Akilandeswari Gopalan
Sent: Wednesday, March 29, 2017 9:38 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] protein precipitation reg
Dear all,
I am a PhD student doing structural
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
dialysi
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