Gel filtration vs gradient

[Dave Parcej]

Hi Pierre,
 this is actually exactly what you would expect if you have a reasonable
amount of detergent bound to your protein (as of course you will). In the
SG experiment the distance migrated depends, among other things, on the
buoyant density of your particle. Since the partial specific volume of
most detergents is in the 0.93ml/g range, while protein is around 0.7ml/g,
the buoyancy of your protein-detergent complex will be higher and it would
therefore migrate less distance into the gradient than a non-detergent
binding protein of the same size. Conversely, on gel-filtration, the
protein detergent complex will appear much larger because of a greater
stokes radius caused by the bound detergent.
If you want to correct for the bound detergent, you can try gradients on
D2O and H2O.
Hope this helps
Dave


In article <383D4A53.373A6085 at pasteur.fr>, Pierre Rodrigues
<pirod at pasteur.fr> wrote:

> Hello,
> 
> We are trying to look at oligomers of an integral protein. We compared
> gel filtration (by HPLC) to sucrose gradients and obtained opposite
> results: in HPLC, most of the protein was eluted on a 300-400 kDa range
> whereas gradient elution was around monomer size!
> 
> As our protein is an integral membrane protein and has 2/3 of its lenght
> 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 100000xg
> clarification before.
> 
> If anyone has some explanations (even partial!) it would be great...
> 
> Thanks
> Pierre Rodrigues
> Institut Pasteur