I am trying to cleave a His-tag on a protein (~60 kDa) using thrombin cleavage. Although thrombin cleavage reactions are typically carried out at pH 8.4, has anyone had success at pH 7.4? The thrombin stock I have is old and I just want to test right now whether PBS will be a suitable choice since my protein seems to crash out of solution at pH 8.4. I am planning to incubate the reactions overnight to ensure complete cleavage. Thanks.
Hi Shivansh,
You could try a time-course (hour/s :0, 1, 2, 3, 5, 8 and overnight) mini trial cleavage of your fusion protein with thrombin in a PBS buffer. You may need to add calcium for the thrombin activity. So try adding calcium into a PBS buffer without protein to confirm whether calcium phosphate precipitation is formed or not before starting the thrombin cleavage trial.
Briefly centrifuge the thrombin stock before use!
Good luck!
I do overnight and it works. I did in Tris buffer. Just try overnight and check gel
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