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Material Data Sheet
SUMO Conjugation Reaction Buffer Kit
Price: $50.00
SUMO Conjugation Reaction Buffer Kit
Price:
50
Quantity:
1 Kit
Data Sheet:
SUMO conjugation reaction buffer kit contains the optimal buffer formulations for use in assays for the conjugation of the ubiquitin-like modifiers, SUMO-1 (UL-712), SUMO-2 (UL- 752), and SUMO-3 (UL-762), to protein substrates in vitro. These SUMO proteins are conjugated to a variety of proteins in the presence of UbcH9 (E2-645) and SAE1/SAE2 (human) (E-315). This kit can supplement the SUMO conjugation kits (K-712, K-715, K-720).
Product Information
Stock:
1. 1ml 10X SUMO Reaction Buffer2. 100μl 10X Mg-ATP3. 1ml 50X E1 Stop Buffer4. 1ml 5X Non-reducing gel loading buffer
Background:
The ubiquitin-like SUMO-1 is conjugated to a variety of proteins in the presence of UbcH9 and the SUMO E1 activating enzyme (SAE1/SAE2 in human, or Aos1/Uba2p in yeast). The heterodimeric SAE1/SAE2 complex (38 and 70 kDa respectively) uses ATP to adenylate the C-terminal glycine residue of SUMO-1, forming a high-energy thiolester bond with the SAE2 subunit. The second step is the trans-esterification reaction whereby the activated SUMO-1 is transferred to Cys93 of UbcH9. UbcH9 is a member of the E2 family and is homologous to ubiquitin-conjugating enzymes, but is specific for the conjugation of SUMO to a variety of target proteins. This E2 is unusual in that it interacts directly with protein substrates that are modified by sumolyation, and may play a role in substrate recognition. Sumoylated substrates are primarily localized to the nucleus (RanGAP-1,<br>RANBP2, PML, p53, Sp100, HIPK2) but there are also cytosolic substrates (IκBα, GLUT1, GLUT4). SUMO modification has been implicated in functions such as nuclear transport, chromosome segregation and transcriptional regulation, apoptosis and protein function and stability.
Recommended Assay Protocol:
Protocol (20 μL reaction):
1. Add 2 μL reaction buffer (1X final) to appropriate enzyme and substrate mix.
2. Bring the volume to a total of 18 μL with dH2O if needed.
3. Initiate reaction with the addition of 2 μL of Mg-ATP solution (1 mM final).
4. Incubate the reaction at desired temperature and time.
5. Dilute 100X stop buffer in dH2O to 10X and stop reaction by adding 2 μL of 10X stop buffer.
6. Add 4 μL 5X loading buffer for SDS-PAGE/Western Blot. For reducing conditions, add 1mM β-mercaptoethanol or DTT (fresh) and boil for 5 minutes prior to loading on gel. Storage:
Store at -20°C. Avoid multiple freeze/thaw cycles.
Literature
References:
Desterro J.M., et al. (1997) FEBs. Lett. 417:297-300
Okama T., et al. (1999) Biochem. Biophys. Res. Comm. 254:693-698
Pichler A. et al. (2005) Nat. Struct. Mol. Biol. 12:264-269
Rodriguez M.S et al. (2001) J. Biol. Chem. 276:12654-59
Saitoh H. and Hinchey J. (2000) J. Biol. Chem. 275:6252-6258
Seeler J-S. and Dejean A. (2003) Nat. Rev. 4:690-699
Su H-L., et al. (2002) Gene 296:65-73
Tatham M.H., et al. (2001) J. Biol. Chem. 276:35368-35374
Yeh E.T.H., et al. (2000) Gene 248:1-14
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