Hi. I was considering using GTP Agarose Resin for the final clean up step of the purification of a GTPase and was wondering if anyone has had experience using this resin. My main concerns are whether it actually has a decent binding capacity for GTP binding proteins, considering that endogenous GTP/GDP may remain tightly bound during purification, and if an active GTPase would bind without cleaving the GTP off of the resin itself.
I found only a few companies that still carry the resin, but the price for each is very different. The resin from Sigma is fairly cheap and is linked to the resin via ribose hydroxyls, while the resin from Innova Biosciences is more than four times as much but is linked to the resin via the gamma phosphate, which supposively prevents cleavage by contaminating phophatases. Considering that my protein should be relatively pure during this purification step, I was wondering whether or not GTP cleavage of the resin by the GTPase and loss of binding to the column would be a problem if using the Sigma resin. If anyone has any other information about purification using this resin, such as resin binding capacity, an effective protocol with relevant buffers, and the lifetime of the resin after regeneration, I would be happy to hear it. Thanks, Matt
