How important is Ca2+ to thrombin cleavage? - (Dec/06/2007 )

Hello,

My question is as in the title. It may be a dumb question for you guys. I am purifying some GST or thioredoxin fused peptide and I need to cut the fusion protein off my peptide from the thrombin cleavage site. Some people insist that 2.5mM Ca should be added to cleavage buffer for the cleavage reaction by thrombin, while some other people don't add Ca2+ and cleavage seems still to work. It confuses me. How important is Ca2+ to the thrombin cleavage? Thanks,

according to a sigma data sheet, thrombin does not require any divalent cations for activity.

Thanks, mdfenko. I thought so. I will try the cleavage without Ca2+ for my next purification.