protein purification!

Dear all,

We are now trying to purify a recombinant protein (his-tag) produced
in E. coli. This protein (inclusion body) is only soluble in high
salt with low pH (4,5). (we usually dissolve it in 8M guanidine at pH
4,5). The protein was purified using his-tag column, but there are
still two extra bands after column purification. We have produced
antiserum in rabbits using the column-purified protein and the
antiserum is going to be purified by an affirnity column, which will
be performed in a commercial company. For this purpose, we need to
produce pure protein so that the commany can use it as a kind of
positive control. We want to elute the protein band from a SDS-PAGE
gel. It has been difficult due to the insolubility of the protein in
most solutions. We tried a commercial available kit to dissolve the
gel, but the gel was not completely dissolved and no protein was
found in the supernatant. Do you have any experience in dissolving
the gel while maintaining the protein's structure unchanged? Any help
is highly appreciated.


Lihua

In article <[Only registered users see links. ].net> , Li-Hua Zhu <[Only registered users see links. ].se> wrote:

What exactly is the affinity column going to be??? If the protein
is misfolded/denatured, the value of such an affinity step is
questionable at best - antibodies against short epitopes will be
selected but also a lot of non-specific garbage will be "purified"
owing to hydrophobic interactions.


You will be better off trying to find conditions that produce partial
refolding of your protein and purifying it. This renatured stuff should be
coupled to the insoluble support. There is tons of literature on the subject
and every other company sells a refolding kit that is a lot better than
other refoldng kits :-)

DK