Also if anyone has any good protocols which can serve as a template for such a large protein complex that would be great.
cheers
Luke
Since the density gradient method is intended to let the macromolecule reach its equilibrium buoyant density, the spins should be long enough to achieve this. I have always done overnight spins, 18-24 hours.
Dear Luke, Adam is absolutely right for the time scale of the experiment. But the most important point is also the centrifugation speed to apply, so that you effectively reach an equilibrium without pelleting all of the protein (as in sedimentation velocity experiments). The right speed to apply is dependant on your protein complex mass, and usually for good results it is important to make different runs at different speeds.
Please, see this paper for more informations :
Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium. Methods Cell Biol. 2008; 84: 143–179.
Dear Yann
Thank you very much for your contribution, greatly appreciated indeed.
I will review the paper.
cheers
Luke
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