AOS1+UBA2 protein (ab3804)

Product Name 

AOS1+UBA2 protein

Product type 

Proteins and Peptides

Specificity 

The final fraction of enzyme contains two single polypeptide bands of 38 kDa and 80 kDa.

Tested applications 
(see key)

FuncS, WB

Application notes 
(see key)

Recommended dilutions
Sumoylation assays are carried out in a final volume of 20 µl in reaction buffer (20 mM Hepes pH 7.5, 5mM MgCl2, 2mM ATP). We would recommend adding fresh 2mM ATP to be sure that sufficient energy is supplied.

Uba 2 contains his-rich regions which might cross-react with antibodies against the 6x-His epitope tag.

During western analysis, Uba 2 at 80 kDa might be shown.

Research areas 

Cell Biology >> Proteolysis / Ubiquitin >> Proteasome / Ubiquitin >> Sumo

Relevance 

E1-SUMO protein is a heterodimer consisted of human AOS1 and UBA2. The dimeric enzyme acts as a E1 ligase for SUMO1, SUMO2, SUMO3, and probably SUMO4. It mediates ATP-dependent activation of SUMO proteins and formation of a thioester with a conserved cysteine residue on SAE2.

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.

Storage buffer 

10 mM Tris-Cl, pH 7.5, 100 mM NaCl, 100 mM imidazole, 0.5 mM PMSF, 1 mM DTT, and 10 % glycerol

Material safety datasheet (MSDS) for this product:
Glycerol MSDS

Purification notes 

The Sumo E1 protein is a heterodimer consisting of human Aos 1 and Uba 2. Both the Aos 1 and Uba 2 are co-expressed in a baculovirus expression system, prepared from freshly extracted cell lysates, and purified to homogeneity. The final fraction of Aos 1 and Uba 2 is an affinity column pool.

Form 

Liquid

Concentration 

0.075 mg/ml

Storage instructions 

Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.

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