problem with gel filtration - (Feb/16/2006 )
I try to purify a membrane protein by gel filtration.
I do first a Ni2+-NTA chromatography, and I get the protein quite pure but absolutely not homogen, as there is in the elution sample monomers, dimers, trimers, tetramers and so on.
The gel filtration that I do afterwards does not separate at all the different oligomers although tehre have ver different size ( monomer 30 kDA, dimer 60 kDa and so on).
All oligomers are eluted in the same pic, coming off in the same collected fractions.
I really do not know how to separate them.
Did anybody already experienced that ? Does it mean all oligomers are bound together ? Is there any possibility to avoid this phenomen ?
Maybe column is too short? Or - too much protein in injected sample?
Besides, what gel are you using?