Hi Matthew, Most GTPases required Magnesium to hydrolyze so you maybe able to reduce this by working in presence of EDTA and absence of magnesium. This may promote removal of traces of GTP or GDP ( from previous experience with SRP GTPases I would be more worried about residual GDP). Having EDTA and no magnesium during purification helps. If your GTPase has a very low basal GTPase activity ( and some do as they require a cognate GAP to really get in the mood to hydrolyze) this might be enough to minimize hydrolysis on this resin.
Good luck, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu On Mon, Jun 13, 2011 at 12:21 PM, Matthew Bratkowski <mab...@cornell.edu> wrote: > 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 >
