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
Dear Praveen,
Here's the summary I give to students when I introduce them to the wonders of
protein purification. It's a bit long (so ... not really a summary, LOL) but
some of it might help you if you're new to this.
Many people don't use removable His-tags as these tags are quite small an
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
