thrombin cleavage and triton? - protein purification (Sep/11/2006 )
I am currently trying to purify a 20kDa protein with both a HIS and GST tag. My extraction buffer contains 1% triton and I would like to know if I can perform a cleavage of the GST tag with thrombin at this concentration. Other material has noted that 0.1% triton is acceptable. Has anyone tried more than 0.1%?
In addition I have imidazole included in my extraction buffer (for the HIS Ni-column). Any recommendations on the best method of purification?
Thanks for your help
If nobody else comes up with a definite idea, you might just have to do the digestion with thrombin at a range of sample dilutions, from neat (1% Triton) to 1/10 (0.1% Triton).
And as long as the imidazole doesn't inhibit the thrombin, don't worry about it until you get to the final purification steps, which, I presume, will be chromatography...
Yes you need to clean up your protein from triton before applying to thrombin. An excellent way to go around this problem without minimal loss of protein concentration, you can use ProteoSpin detergent clean-up kit (http://www.norgenbiotek.com/index.php?id=protkdeter)
Please do not hesiate to contcat me for further infroamtion!