Hi Zhen -
Superdex is known to have some ion-exchange characteristics, so that it
can weakly interact with some proteins. This is why the manufacturer
recommends including a certain amount of salt in the running buffer. I
have had the same experience with a few proteins, including one that
came off the column well after the salt peak! (The protein was very
clean after this step; all other proteins had eluted earlier.)
As others have said, you can't rely on molecular weight calibrations in
this case, but this behavior alone is no reason to think that the
protein is misfolded or otherwise badly behaved. If you don't like the
late elution, try increasing the salt concentration of your running
buffer to 250 or even 500 mM. You'll probably need to exchange the
eluted protein back into a low-salt buffer for your next steps (e.g.
crystallization) if you do this.
- Matt
On 6/20/13 3:09 PM, Zhang, Zhen wrote:
Dear all,
I just observed a puzzling phenomenon when purifying a refolded protein with
size exclusion chromatography. The protein was solubilized by 8M Urea and
refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and
is expected to be a trimer. The puzzling part is the protein after refolding
always eluted at 18ml from the superdex S200 column (10/300), which is
calculated to be 5KDal by standard. However, the fractions appear to be at
40KDal with SDS PAGE and the protein is functional in term of in vitro binding
to the protein-specific monoclonal antibody. I could not explain the
observation and I am wondering if anyone has the similar experience or has an
opinion on this. Any comments are welcome.
Thanks.
Zhen
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