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Product Name
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UBE2I / UBC9 protein
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Product type
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Proteins and Peptides
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Specificity
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The final fraction of enzyme contains a single polypeptide band of 18 kDa.
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Tested applications
(see key)
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FuncS, WB
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Research areas
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Cell Biology >> Proteolysis / Ubiquitin >> Proteasome / Ubiquitin >> Ubiquitin E2s and E3s >> Ubiquitin E2 Enzymes
Cell Biology >> Proteolysis / Ubiquitin >> Proteasome / Ubiquitin >> Sumo
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Relevance
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The modification of proteins with ubiquitin is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation. Ubiquitination involves at least three classes of enzymes: ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s. This gene encodes a member of the E2 ubiquitin-conjugating enzyme family. Four alternatively spliced transcript variants encoding the same protein have been found for this gene.
Catalyzes the covalent attachment of ubiquitin-like protein SUMO-1 to other proteins. Forms a tight complex in association with two RAN GTPase interacting proteins, RANGAP1 and RANBP2. Interacts with E1A and E2A. Interacts with HIPK1 and HIPK2. Interacts with SIAH1 and PARP.
Expressed in heart, skeletal muscle, pancreas, kidney, liver, lung, placenta and brain. Also expressed in testis and thymus.
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.
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Storage buffer
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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
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Purification notes
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Human Ubc 9 (Ubiquitin-like protein SUMO-1 conjugating enzyme) is produced from an E. coli expression system, prepared from freshly extracted cell lysates, and purified to homogeneity. The final fraction of the Ubc 9 is an affinity column pool.
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Form
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Liquid
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Concentration
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0.500 mg/ml
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Storage instructions
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Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
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Expression system
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E. coli
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At Abcam, we have one centralized database to hold all of our product information, so that everything we know about this UBE2I / UBC9 protein is on this datasheet. But please do contact us if you would like any reassurance! |
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See below for UBE2I / UBC9 protein images, references, products related to ab3803 and other tools.
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UBE2I / UBC9 protein images:
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 Sumoylation assay
Left Lane: Topo I protein (Topo I fragment: ab3828)
Middle Lane: Sumo 1 sumoylated Topo I (Sumo 1: ab3801; Topo I fragment: ab3828; EI: ab3804; EII: ab3803; Buffer: ab3827)
Right Lane: Sumo 3 sumoylated Topo I (Sumo 3: ab3802; Topo I fragment: ab3828; EI: ab3804; EII: ab3803; Buffer: ab3827)
Note: Topo I is S35-Met labeled.
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Topo I sumoylation
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).
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Western Blot: 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.
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Search PubMed (MEDLINE) for references to UBE2I / UBC9
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