Sugden lab,McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School
http://mcardle.oncology.wisc.edu/sugden/Protocols/html files/Thrombin cleavage.htm
Thrombin cleavage
(using thrombin produced by Amersham)
1. Thrombin cleavage of eluted fusion protein bound to Sepharose
* Mix 50 μl of thrombin (< 10 cleavage U/ml) solution and 950 μl of 1 x PBS for each ml of Glutathione Sepharose bed volume
Add thrombin protease mixture to Glutathione Sepharose pellet
Gently shake or rotate the suspension at r.t. for 2-16 h
Centrifuge the suspension at 500 x g for 5’ to pellet the beads and carefully transfer the eluted fraction to a clean tube.
2. Thrombin cleavage of eluted fusion protein.
Add 10 μl of thrombin solution (10 cleavage units) per mg fusion protein. If the amount of fusion protein in the eluate has not been determined, add 80 μl (80 U) of thrombin for each ml of Glutathione Sepharose bed volume from with the fusion protein was eluted.
Mix gently and incubate at r.t. (22-25C) for 2-16 h.
Once digestion is complete, GST can be removed by first removing glutathione by extensive dialysis (2,000 vol/ml) against 1 x PBS followed by batch purification.
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