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Protein elution from GST-fusion protein complex


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#1 Ana R Santos

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Posted 29 September 2010 - 06:49 AM

Hi there,

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... ;)

#2 madrius1

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Posted 30 September 2010 - 07:24 AM

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.

#3 Ana R Santos

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Posted 30 September 2010 - 01:27 PM

I have used BS3 to crosslink..

I am going to try Urea, 7M.. it very harsh..but i hope i can get something out.

Thanks*

#4 laurequillo

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Posted 30 September 2010 - 07:30 PM

You can boil them in sds sample buffer
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