Sumoylation Control Kit

(Catalog # K010 )

 Kit Contents:

• 30 reactions of human Aos1/Uba2 (E I), active (Cat# P006).

• 30 reactions of human UBC9 (E II), active (Cat# P011).

• 30 reactions of human SUMO I, active (Cat# P012).

• Human p53 marker (cat# B011, G S T- p53 protein (83 KD) for sumoylation purpose only without enzymatic activity, 20µl per tube, good for 20 standard assays).

• SUMO I antibody (cat# B013, 20 µl, 1000x dilution for western blot, good for 20 standard assays).

• G S T antibody (cat# B012, 20 µl, 1000x dilution for western blot, good for 20 standard assays).

• 10X sumoylation reaction buffer (cat# B010, 200 µl, good for 100 standard assays).

p53 Sumoylation Control Protocol:

1.      Add 1 µl p53 marker (Cat# B011) for each reaction.

2.      In each reaction, add 1µl UBC9 (E2) to substrate first, then 1µl Sumo-1, 2µl 10x reaction buffer (Cat# B006), 1µl Aos1/Uba2 (E1). Finally, add H₂O to bring up to 20µl  (we would recommend to add fresh 2mM ATP to be sure that sufficient energy is supplied).

3.      The best reaction concentration of  sumoylation proteins is as following:

4.      Use 1000x dilution of supplied G S T antibody in 10 ml (Cat# B012) for western blot. One sumoylated band shall be seen on the gel.

Western blots (7.5% gel) of sumoylated G S T-p53 fusion protein (cat# B011) with G S T antibody (cat# B012). Lane 1: unsumoylated; Lane 2: sumoylated G S T-p53.

Western blots (7.5% gel) of sumoylated G S T-p53 fusion protein (cat# B011) with SUMO 1 antibody (cat# B013). Lane 1: unsumoylated; Lane 2: sumoylated G S T-p53.

1.      Prepare the suitable purified substrate protein.

2.      In each reaction, add 1µl UBC9 (E2) to substrate first, then 1µl Sumo-1, 2µl 10x reaction buffer, 1µl Aos1/Uba2 (E1). Finally, add H₂O to bring up to 20µl (we would recommend to add fresh 2mM ATP to be sure that sufficient energy is supplied).

3.      The best reaction concentration of  proteins is as following:

Legal consideration: FOR RESEARCH USE ONLY. 

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