Gel filtration vs gradient
nicholas_theodorakis at urmc.rochester.edu
Thu Nov 25 21:06:31 EST 1999
In article <383D4A53.373A6085 at pasteur.fr>, Pierre Rodrigues
<pirod at pasteur.fr> wrote:
> We are trying to look at oligomers of an integral protein. We
> gel filtration (by HPLC) to sucrose gradients and obtained opposite
> results: in HPLC, most of the protein was eluted on a 300-400 kDa
> whereas gradient elution was around monomer size!
> As our protein is an integral membrane protein and has 2/3 of its
> hydrophobic, could there be lipids still interacting with it and
> make it
> float on gradients?
> We did this experiments on Triton x-100 and CHAPS and did a
> clarification before.
> If anyone has some explanations (even partial!) it would be
> Pierre Rodrigues
> Institut Pasteur
What is the micelle molecualr weight of Triton? Could you actually be
looking at the size of your protein in a Triton micelle?
Alternatively, the usual explanation for a protein behaving larger than
it "ought" to during gel filtration is that is highly elongated, and
sweeps out a relatively large volume during chromatography.
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