Protein elution from GST-fusion protein complex - (Sep/29/2010 )
I am using a GST-fusion protein to pulldown ubiquitinated proteins. My final goal is to cut off the ubiquitinated proteins from the GST-fusion protein complex.. so then o have a pure sample of ubiquitinated proteins to be analyzed.
I have cross-linked the GST-fusion protein to agarose beads and then try several elution strategies, including, 0.2M Glycine ph2.5 (1min, 5min, 30min and 1h); Urea; adding detergents or salt.... but none of those buffers semms to work, as i still get the ubiquitinate dproteins attached to the beads.. meaning that the elution was not OK. In the GST control i have signal.. so, it seems specific.
I hope that anyone can help.. this is my last experiment...
Have you tried eluting with SDS?
Also, what kind of crosslinking did you do? If you used formaldehyde, you can reverse the crosslink by incubating your samples 5 min at 95 degrees.
I have used BS3 to crosslink..
I am going to try Urea, 7M.. it very harsh..but i hope i can get something out.
You can boil them in sds sample buffer