How much thrombin for GST fusions ?

Curt Ashendel ashendel at aclcb.purdue.edu
Wed Feb 19 09:02:41 EST 1997


On Tue, 18 Feb 1997 23:15:21 +0000, Tom Haenel   
<tehaenel at cyllene.uwa.edu.au> posted to bionet.molbio.methds-reagnts:

>Does anyone know the number of NIH units of thrombin to use per mole of
>fusion protein ?

The recommendation I got from another lab (I am about to do this 
myself today) is 1 to 5 units per mg of protein (for fusions of 40 
to 100KD), room temp, one hour. But this varies with the purity of the 
thrombin (they use high purity stuff) and whether or not the fusion 
protein has a glycine kinker region at the fusion point, which 
enhances the rate of cleavage (the above number is for fusions with 
the kinker).

It probably also varies with the fusion protein structure and 
concentration. The lab I talked to recommended a small scale (pilot) 
reaction with timed removal of aliquots for SDS-PAGE analysis.



Curt Ashendel
Purdue University, West Lafayette, IN
ashendel at purdue.edu



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