How much thrombin for GST fusions ?
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
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.
Purdue University, West Lafayette, IN
ashendel at purdue.edu
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