Have you investigated ion exchange chromatography?

Yes, I did, but unfortunately I only got that my complex broke up. The subunits are eluted separately, at different salt concentrations.

Hi Patrizia,

I don't think the resolution of glycerol gradient sedimentation is high enough to separate your two complex.

I would suggest you to optimize the ion exchange purification. You can screen different buffers with different low concentration salt (eg. 20-100mM), to get a condition in which your complexes are stable and can be separated.

Lin

The complex might have broken up, but have you tried to assemble them again? Separating the subunits and then reassembling them might be a good idea, unless these two complexes contain the same subunits but in different ratios.

@Jackie: yes, I tried to assemble them again, but I get the same situation, and I think you're right, they could be two complexes with different ratios of the same subunits.

@Lin: thank you for your suggestion. I think I will explore more conditions with ion exchange, probably I'll try also with a different column...I always used MonoQ, but probably it could be worth to try with a MonoS, too... Hope It works.

Thank you all for your kind reply!

With our eXpert software I tried to simulate the conditions in silico. Assuming the bigger protein relates to a sedimentation coefficient of 10 and the lighter of 9 (calculated as "globular proteins", density 1.3 also by the eXpert software) and using a gradient of 5 - 20% sucrose there could be a chance to get a more or less proper separation (see the document) using 4 ml of volume, 60.000 rpm and 12 hours of run.

Maybe I could help you... Please let me know if you tried it and how it worked.