Re: [ccp4bb] Off-topic Thrombin cleavage

2012-05-24 Thread Seema Nath 
I have used both Novagen thrombin  lyophilized thrombin of 
BDBioscience(dissolved in 10X thrombin dilution buffer provided by Novagen) in 
both 'thrombin cleavge buffer'  others (HEPES pH 7.0, Bicine pH 9.0, MOPS pH 
7.8) without adding calcium chloride - all the conditions permitted the cleavge 
process, but addition of DTT/ BMe slowed the process

[ccp4bb] Off-topic Thrombin cleavage

2012-05-23 Thread Yuri Pompeu 
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

Re: [ccp4bb] Off-topic Thrombin cleavage

2012-05-23 Thread Debasish Chattopadhyay 
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