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... ;)
Thanjs Miguel! Indeed i have crosslinked the GST-fusion proteins to agarose beads. The idea of eluting the proteins of interest is then to resolve them by 2D gels.. and thats why i want to get rid of GST.. because in a preliminar analysis i got tons of GST contaminants.
What i don't understand is why glycine (and even UREA) is not enought to elute the ub-proteins.
Thanks anyway!!
Rita*
if you successfully x-linked your GST-baits onto the resin, even the SDS sample buffer boiling should not elute any of the covalently linked GST-baits. (in response to an earlier reply)
I would guess the 0.2M Glycine 2.5 wash should be able to elute your ubiquitinated prey proteins. Have you tried washing it with multiple column volume of glycine? We usually do 5x CV of glycine elution, and concentrate the peak fractions.
The other possibility is, without knowing the source of your prey protein, your prey protein could be unfolded or misfolded, so that they may form unsolvable aggregates, which non-specifically collapse onto your resin. Therefore, the glycine elution was not able to elute it off. If this is the case, you can try a different expression source of your prey or change your extraction method/buffer recipe.
Good luck!
Lu
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods