Description:
This assay kit contains reagents for routine detection of sumoylation activity. Sumoylation marker (human Topoisomerase I protein) is included to allow clear detection of the reaction. This assay has been shown to work with crude extracts. Human E I, active (Cat# P001; P006), Human E II, active (Cat# P002; P011), and Human SUMO I, active (Cat# P003; P012) are strongly recommended to be used along with this kit.

K007 Kit Contains:

• SAE I and SAE II (Human E I)  (Cat# P006)
• UBC9 (Human E II)  (Cat# P011)
• SUMO I (active form).  (Cat# P012)
• Human DNA Topoisomerase I fragment.  (Cat# B007)
• Anti-Topoisomerase I antibody. (Cat# AP006)  (Lyophilized antybody should be reconstituted with 100 ul of H₂O before use.)
• 10X sumoylation reaction buffer. (Cat# B006) (10x buffer contains 200 mM Hepes pH 7.5, 50 mM MgCl₂, and 20 mM ATP.)

Assay Conditions:
Sumoylation assays are carried out in a final volume of 20 ul in reaction buffer containing 20 mM Hepes pH 7.5, 5mM MgCl₂, 2mM ATP. We would recommend to add fresh 2mM ATP to be sure that sufficient energy is supplied.

Topoisomerase I Sumoylation Control Protocol:

1. Add 2 ul of 50 kd Topoisomerase I-fragment (Cat# B007) as substrate for each reaction.
2. In each reaction, add 2 ul of E2 to substrate first, then 2 ul of Sumo-1, 2ul of 10x reaction buffer (Cat# B006), 2ul of E1 and 2 ul of 20 mM ATP.  Finally, add H₂O to bring up to a total volume of 20 ul .
3. The best reaction concentration of LAE sumoylation proteins is as following:
   • E I:  7.5 ug/ml
   • E II:  50 ug/ml
   • SUMO-1:   50 ug/ml
         The recommended condition for control assay is 37 ^oC for 30~60 minutes.
   
   
4. Use 1000x dilution of supplied Topoisomerase I antibody in 10 ml (Cat# AP006) for western blot. Four major sumoylated bands shall be detected.

Western blot (12.5% gel) of sumoylated 50 Kd Topo-I protein (cat# B007) with Topo-I antibody (cat# AP006).  Lane 1: sumoylated 50Kd Topo-I fragment; Lane 2: unsumoylated 50Kd Topo-I fragment.