It depends on the accessibility of the cut site when bound to the column, but
we have a lot of luck with doing an on-column TEV cut of his-tagged proteins.
We load the protein on a cobalt column, mix in his-tagged TEV, and cut
overnight. In the morning the cut protein just flows off, the tag a
In continuation of this thread, what is the success rate of separating TEV
and MBP tag from my desired protein after TEV cleavage using
Hydrophobic/Ion exchange column?
I have also read TEV is less active in a high concentration of Imidazole,
so dialysis before adding TEV may work better but then r
Hi Liuqing,
The scheme you suggested (or heard) of using Ion-exchange at the end can be
useful to separate protein with same net charge but different conformations.
Ciao
On Fri, Nov 17, 2017 at 1:49 PM, Gianluca Cioci
wrote:
> Dear All
>
> The ion exchange has the great advantage ,over other t
Dear All
The ion exchange has the great advantage ,over other tecniques, to
concentrate the protein.
Histrap+sec is a big classic in protein purification but times it is worth
considering other schemes. Why doing a SEC? It is for removing aggregates
or a contaminant ? It the latter case probably
Hi Liuqing,
I would not recommend SEC. SEC does not give that great of a separation
unless your contaminant is greatly different in size. Instead of SEC, you
might want to consider hydrophobic interactions chromatography (HIC). You
can add your ammonium sulfate directly to your eluted protein f
Hi Liuqing,
I agree with what Pascal has already written and can add something from my
experience.
In one case I needed to purify a protein using gelfiltration to control the
exact composition of the buffer before preparing for the ion exchange.
The protein was only stable in high salt buffers. D
Hi liuqing,
It is more usual to finish with a sec because you control the composition
of the final conditioning buffer of your sample and remove any aggregates.
We personally favor the following sequence
iMac - desalt - iex if necessary - sec
Desalt meaning one of those small Pd10 like column th
Hello everyone!
I have listened someone suggested that, first use affinity chromatography
(Ni-NTA), then use SEC (superdex200 increase), and finally used ion exchange
(monoQ), to purified protein, which will be used to crystallization.
My question is why the monoQ used in the finally step
