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Mild, non SDS based elution conditions for disrupting protein-protein interactio - (Nov/28/2006 )


I have been trying elute my proteins bound to immobilized protein-bead complex using 7M urea, 2M thiourea, 4% CHAPs and 30 mM TRis (pH 8.8) by end-to-end rocking for 2 hours at 4degrees. I then precipitate my proteins using the 2D cleanup kit from GE Healthcare and solubilize the proteins in 50 mM tRis and 0.1% SDS for ICAT analysis. However I think I am eluting very little amount of proteins off of the beads.
Can someone help me with this problem? sad.gif

Thank you for your inputs in advance.



Have you measured how much was bound before elution?


QUOTE (WAstate @ Nov 28 2006, 11:39 AM)
Have you measured how much was bound before elution?

Thank you for the reply.
I did not measure the protein concentration before the elution. I am not sure how to measure the concentrations of proteins bound to beads ?
Secondly, when I tried to precipitate proteins from my eluate, I could not even see the pellet.