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
I personally like TRIS for the first few steps of purification and then change
to something else during my last dialysis step. I mostly work with bacteria and
they often produce lysates that have pH's that are too acidic for good nickel
affinity chromatography, which is why I use 100 mM TRIS pH
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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
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 stud
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