gel filtration vs gradient

H.V.Taylor harold.taylor.remove-this at delete-this.uni-tuebingen.de
Thu Nov 25 15:00:48 EST 1999


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




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