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Cat. No.: SUMO-1k (for 1,000U)

Cat. No.: SUMO-5k (for 5,000U)

Description

SUMO protease specifically cleaves the SUMO moiety in a ‘scarless’ manner. SUMO protease recognizes the tertiary structure of the Ubiquitin-like SUMO domain and hydrolyzes the peptide bond in the x–Gly–Gly–x sequence after the Gly-Gly bond, at the C-terminus of the SUMO domain. In addition to the cleavage of natural SUMO-modified proteins, SUMO protease is used to cleave recombinant SUMO fusion proteins. The SUMO domain is a known solubility-enhancing fusion tag used in recombinant protein expression. A histidine tag can also be added at the N-terminus of the SUMO domain as a purification tag. This SUMO protease product carries a 6x histidine tag. Thus it can be easily removed together with the cleaved SUMO domain, following the digestion reaction
SUMO protease is active over a wide range of temperatures (2–30 °C), ionic strengths (0–400 mM NaCl), and pH ranges (6–8.5). However, its activity may vary depending on the substrate and conditions. Researchers will need to optimize their specific reaction conditions. As an initial suggestion, 20 units of SUMO protease can be used per mg 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. If necessary, the amount of SUMO protease can then be adjusted. SUMO protease works better in the presence of reducing agents, e.g., 0.5–2 mM DTT. DTT in the reaction mixture can significantly enhance cleavage efficiency, especially during longer incubations

Unite definition

In 1 x SUMO Buffer-Salt (50 mM Tris-HCl, pH 8.0, 0.2% NP-40, 1 mM DTT), the amount of enzyme required to cleave >85% of the 2 µg control substrate at 30 °C for 1 h is defined as an active unit.

Storage

SUMO Protease can be stored for 2 years at -80 °C for a long time or for 6 months at -20 °C after sub-packaged to avoid repeated freezing and thawing. 10 x SUMO Buffer+/-Salt can be stored at -20 °C.

Protocol

If the protein is thermally unstable, incubate at 4 °C for a longer time or increase the amount of enzyme used.

1. Set up the following reaction system in EP tube:
2. After mixing the system, incubate at 30 °C. Take 30 μl solution at 1, 2, 4, and 6 h respectively, and place it in a separate EP tube.
3. Add 20 μl 2 x SDS Loading Buffer to the above EP tube and place it at -20 °C.
4. Take 30 μl sample for SDA-PAGE analysis.

Note

• 10 x SUMO Buffer + Salt: 500 mM Tris-HCl, pH 8.0, 2% Igepal (NP-40), 1.5 M NaCl, 10 mM DTT. 10 x SUMO Buffer-Salt: 500 mM Tris-HCl, pH 8.0, 2% Igepal (NP-40), 10 mM DTT. Please select the appropriate buffer according to the experiments.
• To achieve the best digestion result, please ensure that the recombinant protein is partially or completely purified. 
• For different fusion proteins, the concentration of NaCl required by SUMO protease will vary and can be adjusted between 0-300 mM.