I tried a pH of Tris 8.8 pH but after TEV cleavage, I find there is less cleavage and not much desired protein. Which buffer should be used so that no changes in between would be done to prevent protein precipitation?
According to information from InVitrogen, the standard condition for TEV cleavage is 50 mM Tris (pH 8.0), 0.4 mM EDTA, 1 mM DTT. If 8.0 is the optimal pH, then its activity may be somewhat lower at pH 8.8, but I would expect it still to be active. It is not unusual to get poor cleavage with TEV, probably because the cleavage site is not well exposed. That assumes the correct cleavage site is present: Glu-Asn-Leu-Tyr-Phe-Gln/Gly.
Do you actually know for a fact that your protein will precipitate if you perform the Tev treatment under the conditions recommended for optima Tev protease activity ? Like Adam says, it is likely that the Tev site of your protein is not well exposed. Before performing a preparative cleavage on a latge batch of protein substrate, it is advisable to perform an analytical titration with increasing amounts of Tev protease to determine the optimal amount of protease. This can be done in small aliquots of 20 µl containing 5 µg of target protein and checking the outcome of the reaction by SDS PAGE
Thanks Sir, should I use a buffer of Tris, pH 8 or 6.8 to be on a safer side? My fifty percent of the protein is cut only and I fear that after cleAVAGE I MIGHT NOT lose the protein as it becomes close to pI
Dear Swati,
I would try to find the optimal concentration of Tev protease at pH 8, but with some salt in the buffer (100 mM NaCl) as outlined above. Then, I would process a slightly larger batch (50 µl), centrifuge the reaction mixture (e.g. 20 min in a microcentrifuge) and analyze the pellet and the supernatant by SDS-PAGE. Tev protease doesn't like high salt concentrations, but I have performed Tev cleavage with as much as 0.5 M NaCl.
With proteins, there is no alternative but trial and error.
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