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

Hi,


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?

Thank you for your inputs in advance.

H.S

---

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.

HS

---