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Our Abpromise guarantee covers the use of ab3803 in the following tested applications.
This product can be used as part of an assay for sumoylation activity. Human Aos 1 + Uba 2 (ab3804), Ubc 9 (ab3803) and Sumo 1 (ab3801) can be used to promote in vitro sumoylation of a sumoylation marker (human Topoisomerase I protein fragment) (ab3828). The reaction products can be detected using our Sumo 1 (ab3819 and ab3824) and Topoisomerase I (ab3825) antibodies. Sumoylation assays are carried out in a final volume of 20µl in reaction conditions (20 mM Hepes pH 7.5, 5mM MgCl2, 2mM ATP). Sumoylation Protocol: 1. Prepare a suitable purified substrate protein. (For the control, use 2µl Topoisomerase I marker for each reaction.) 2. In each reaction, add 4µl E2 to substrate first, then 2µl Sumo 1, 2µl 10x reaction buffer, 2µl E1. Finally, add H2O to bring up to 20µl. We would recommend adding fresh 2mM ATP to be sure that sufficient energy is supplied. 3. The best reaction concentration of proteins is as following: Aos 1 + Uba 2: 7.5µg/ml. Ubc 9: 50µg/ml. SUMO 1: 50µg/ml. For the control assay we recommend running the assay at 37ºC for 30-60 minutes. 4. Detect the reaction products by Western blot using a suitable antibody. For the control reaction use 1/1000 dilution of the supplied Topoisomerase I antibody. Four sumoylated bands should be seen on the gel for the control reaction. This assay has been shown to work with crude extracts. Be aware that Uba 2 contains his-rich regions which might cross-react with antibodies against the 6x-His epitope tag. During western analysis with anti-6x-His antibodies, Uba 2 at 80 kDa might be shown.The final fraction of enzyme contains a single polypeptide band of 18 kDa.
Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
10 mM Tris-Cl, pH 7.5, 100 mM NaCl, 100 mM imidazole, 0.5 mM PMSF, 1 mM DTT, and 10 % glycerol
Left: Human topo I (S35Met-labeled) control.
Right: Sumoylated human topo I (S35Met-labeled)
(7.5 µg/ml E1, 50 µg/ml E2, 50 µg/ml sumo1, 37ºC 30min).
Extracts prepared from CEF cells not transfected (-) or transfected (+) with wild-type Src were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to nitrocellulose. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with 1.0 µg/mL anti-Src pan antibody. After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG alkaline phosphatase and the signal was detected using the Tropix WesternStar method.
ab3803 has not yet been referenced specifically in any publications.