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:[log in to unmask]] On Behalf Of Yuri Pompeu
Sent: Wednesday, May 23, 2012 12:23 PM
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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
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