Thrombin works pretty good without any added calcium. We routinely added thrombin to whatever buffer the protein is in provided it doesn't have a lot of DTT. Some beta-mercaptoethanol is alright. What is the source of your thrombin?
-----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yuri Pompeu Sent: Wednesday, May 23, 2012 12:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic Thrombin cleavage Dear community, I am trying to cleave a hexaHis tag from my protein prior to crystallization. As I was setting up my digestion, my protein started to precipitate as soon as I added the recommended thrombin buffer. My question is, if anyone has encountered this, how well does it cleave without thrombin buffer? or even, without the CaCl2? Any other buffer/conditions known to work? thanks in advance
