Protein elution from GST-fusion protein complex
Posted 29 September 2010 - 06:49 AM
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...
Posted 30 September 2010 - 07:24 AM
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.
Posted 30 September 2010 - 01:27 PM
I am going to try Urea, 7M.. it very harsh..but i hope i can get something out.
Posted 30 September 2010 - 07:30 PM
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