Maybe you can this way.
Use Na acetate to elute your protein, then use EDTA to remove the Ni
from your protein, then buffer exchange or dialysis to remove EDTA.
Kevin
On Fri, Mar 2, 2012 at 5:50 AM, Santosh wrote:
> Hi Anita,
> As Artem noted, use of Histrap column at lower pH would not be a
Hi Anita,
As Artem noted, use of Histrap column at lower pH would not be a great idea
and unless you want to elute your protein using pH Step gradient
http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/A960DBAAE1C0C945C1257628001D29BB/$file/28404480AA.pdf
You may also consider using CM (Carb
Classical protein purification by IEX, HIC,
GEC, etc. is apparently a dying art. It is typically quite easy to
purify proteins using non-affinity methods from overexpression
mixtures using an AKTA system. (Gosh, in the old days we used to
purify to homogeneity pro
Proteins with high apparent pi value are often tricky because they tend to
bind anionic substances such as nucleic acids, other proteins, glass, etc.
Conversely given that most proteins have acidic-ish apparent pi its often
worth looking into why a particular protein is basic as it may be a fact of
You might be giving too much importance to the THEORETICAL pI of the protein.
If it's supposed to be well charged at pH 7,4 (only a titration curve, and not
simply knowing the pI will tell you this) and it's still precipitating, the
problem might be due to a bad fold, for instance, or to the la
This pH is generally incompatible with Ni IMAC, sorry :) If you have a
high pI your best bet is to employ ion exchange as primary capture,
specifically SP resin or if you're really lucky - CM resin. There are
only relatively few proteins in E. coli that bind to CM resin at pH 5
and virtually none (
Hi all,
Has anyone used sodium acetate buffer pH (4-5) for purifying histag protein
on Histrap column (AKTA) followed by SEC?
My protein has a pI of 9. I tried pH7.4 but it has precipitation problems.
While doing buffer screening using 24 well hanging drop I found that lower
pI onces are clear, so
