Pierre Rodrigues <pirod at pasteur.fr> wrote:
>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!
>Dear Pierre,
is it possible that sucrose "de-polymerizes" your protein? Try using
sucrose in your HPLC buffers to see if that changes things (though not
too high a concentration as you'll probably get a very high
back-pressure). You might take a look at Schägger's blue native
electrophoresis of membrane proteins with coomassie blue. Here's a
reference:
Human diseases with defects in oxidative phosphorylation. 2. F1F0
ATP-synthase defects in Alzheimer disease revealed by blue native
polyacrylamide gel electrophoresis.
Schagger-H; Ohm-TG
Eur-J-Biochem.1995 Feb 1; 227(3): 916-21.
Hope this helps,
Harold
Physiol. Chem. Institut
Hoppe-Seyler-Str. 4
D-72076 Tübingen
phone: 07071-297-3353
fax: 07071-29-4182
email: harold.taylor at uni-tuebingen.de
