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I'm trying to isolate a ~320kD protein (in oligomeric form) using density gradient ultra centrifugation technique.
Can any one tell me that what gradient (sucrose or glycerol or trehalose or etc) I should use and why ?
Any references regarding the optimization of gradient cutoff for macromolecules will be highly appreciated.

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  • $\begingroup$ Have you tried size exclusion chromatography (SEC)? It's a lot easier to set up, perform, and analyze than gradient centrifugation. $\endgroup$
    – MattDMo
    Commented Jun 17, 2016 at 21:44
  • $\begingroup$ Yes it is. But if you are trying to separate between 240kD and 320kD then it is not proving to be much of a helpful technique as both is coming in void volume in Superdex column. I have tried but not helpful in this scenario. $\endgroup$
    – diffracteD
    Commented Jun 18, 2016 at 6:51
  • $\begingroup$ Have you tried different columns? If your analyte is coming out in the void, try altering the particle size in the column. $\endgroup$
    – MattDMo
    Commented Jun 18, 2016 at 15:54
  • $\begingroup$ Yes, but given the availability of columns it is not being possible to resolve in this range using SEC procedure. That's why I was trying for density ultra centrifugation. $\endgroup$
    – diffracteD
    Commented Jun 18, 2016 at 16:09
  • $\begingroup$ OK, I understand. Unfortunately, the only experience I have with density centrifugation is in purifying white blood cells from whole blood using Ficoll gradients, so I'm afraid I can't help with this. Good luck, though! $\endgroup$
    – MattDMo
    Commented Jun 18, 2016 at 16:11

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