Recombinant SUMO Protease, Yeast

1. SUMO Protease;

2. 10X SUMO Protease Buffer + Salt: 500 mM PB, pH 7.4, 2% Igepal (NP-40), 1.5 M NaCl, 10 mM DTT;

3. 10X SUMO Protease Buffer – Salt: 500 mM PB, pH 7.4, 2% Igepal (NP-40), 10 mM DTT;

1.Add the following to a microcentrifuge tube:

2. Mix and incubate at 30°C, Remove 5μl aliquots at 1, 2, 4, and 6 hours.

3. Analyze by SDS-PAGE.

• Keep the concentration of Imidazole less then 150mM, or the activity of the SUMO Protease can be adversely affected.
• For most fusion proteins, SUMO Protease functions optimally in a reaction mixture containing 150 mM NaCl; however, conditions may be optimized by varying the NaCl concentration from 100 mM to 300 mM. Remember to take into account the contribution of salt from the enzyme and from your substrate. When setting up your cleavage reaction, use the appropriate 10X SUMO Protease Buffer +/- Salt.
• Researchers need to optimize their specific reaction conditions. As an initial suggestion, 20 units of SUMO protease can be used per 40μg of target protein for 1 hour at 30 °C, or overnight at 2–8 °C. The cleavage efficiency can then be estimated by SDS-PAGE.