Why does AcTEV Protease require DTT?
A final concentration of 1 mM DTT is required for the AcTEV protease reaction. The DTT serves as a stabilizer of secondary structure, i.e., it ensures that the enzyme does not undergo oxidation. If you are unable to use DTT due to a column purification after digestion, you can leave it out and still see a successful AcTEV digestion, however we recommend using it.
No rigorous quantitative data for activity in the absence of DTT and EDTA has been collected in-house. Incubations using reaction buffer +/-DTT and +/- EDTA; have been performed, but the products were analyzed only at t=0 and t=2 hr at 30 degrees C. No difference in the amount of product generated +/-DTT or +/-EDTA was noticed, but it is possible that the detailed kinetics in a time course reaction may be a little different.
No rigorous quantitative data for activity in the absence of DTT and EDTA has been collected in-house. Incubations using reaction buffer +/-DTT and +/- EDTA; have been performed, but the products were analyzed only at t=0 and t=2 hr at 30 degrees C. No difference in the amount of product generated +/-DTT or +/-EDTA was noticed, but it is possible that the detailed kinetics in a time course reaction may be a little different.