roysi...@gmail.com
On Monday, December 26, 2016 2:29 PM, Mark J van Raaij
wrote:
if you can figure out what is making your protein precipitate, you could put
something in the collection tubes of the NiNTA column to prevent precipitation.
For example a bit of concentrated buffer to cha
if you can figure out what is making your protein precipitate, you could put
something in the collection tubes of the NiNTA column to prevent precipitation.
For example a bit of concentrated buffer to change the pH, or EDTA.
Another thing, you say your protein is pure after NiNTA, but it may stil
Hi,
Regarding the second publication below on the optimum solubility screen, I was
a new postdoc in Sung-Hou Kim's lab when the last parts of the work and
manuscript was being prepared, and so did quite a few experiments using the
screen in conjunction with DLS.
It is worth trying them out bu
Hi,
although most widely used, Tris-NaCl buffers may be acceptable for many
but unfortunately by far not all proteins.
The solubilty of a protein can be modulated by the use of the proper
anions and cations. This all depends on the pH of the solution and the
pI of the protein.
Consult the
Hey Praveen
There are wonderful advices in all the emails. Having over 8 years
experience in protein purification, i still admire the way Antonio Ariza
has summarized a work flow. You shall need to devise a strategy to optimzie
the purification protocol. I could not resist to ask, why are not usin
Likely, the protein is pH sensitive.
You may chain the anion and cation columns together for protein separation,
then you don't need to use Imidazole.
P.S. Tris pH 7.5 (RT) is ~ pH 8.2 in cold room
On Sat, Dec 24, 2016 at 2:52 AM, Praveen Tripathi <
tripathipraveen2...@gmail.com> wrote:
> Dea
Hi,
Check pI vs pH and try different pH. Can also try room temp purification.
Try lower salt conc. Is it a DNA-binding protein? Also consider adding a ligand
or partner if known. Your 10% glycerol should help to improve solubility but
you can also try 15% glycerol, which should also help in st
een Tripathi
[tripathipraveen2...@gmail.com]
Sent: 24 December 2016 10:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Need suggestion for protein solubility
Dear all,
I am graduate student working on a functional protein which i have cloned in
pET-28a vector for recombinant protein production in E.coli
Elute in batch and remove imidazole immediately :) half the time imidazole
is the enemy.
Artem
www.harkerbio.com
"From gene to sausages."
Artem
On Dec 24, 2016 6:04 AM, "Praveen Tripathi"
wrote:
> Dear all,
> I am graduate student working on a functional protein which i have cloned
> in pET-28
Dear all,
I am graduate student working on a functional protein which i have cloned
in pET-28a vector for recombinant protein production in E.coli expression
system.
The expressed protein is purified on Ni-NTA resins with Imidazole gradient.
Surprisingly, i am getting distinct visible white precipi
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