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Ubiquitinylation kit

Versatile tool for generation of ubiquitin-E2 thioesters.
BML-UW9920-0001 1 Kit 502.00 USD
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  • Excellent tool for generating a range of thioester-linked ubiquitin-conjugated E2 enzymes
  • Biotinylated ubiquitin provided for sensitive detection with streptavidin-linked enzymes
  • Kit provides sufficient material for 50 x 50 µl reactions
For generation of ubiquitin-E2 thioesters for use in ubiquitinylation experiments. This kit provides the means of generating a range of thioester-linked ubiquitin conjugation enzymes (E2s), utilizing the first two steps in the ubiquitin cascade; for use in the transfer of ubiquitin to E3 ligases; and the subsequent ubiquitinylation of target proteins.Biotinylated ubiquitin provided supports thioester formation and high sensitivity detection of ubiquitin conjugates. Suggested uses: Generation of ubiquitin-E2 thioesters for use in ubiquitinylation experiments, Ubiquitinylation of target proteins in the presence of a dedicated E3 ligase, Activation of ubiquitin for thioester conjugation to novel E2 enzymes, Use of cell lysate or crude fractions/preparations as source of E3 ligases to facilitate ubiquitinylation, Substrate (target) independent in vitro ubiquitinylation reactions.
Figure: Western Blot of Thioester Assays (TE +ve/-ve controls) for all E2 conjugating enzymes provided. Procedures as described in “Assay Protocol” section. Biotinylated-ubiquitin-enzyme conjugates were detected by Western Blotting on thioester assays containing A: UbcH1 (Prod. No. BML-UW9020), B: UbcH2 (Prod. No. BML-UW9025), C: UbcH3 (Prod. No. BML-UW8730), D: UbcH5a (Prod. No. BML-UW9050), E: UbcH5b (Prod. No. BML-UW9060), F: UbcH5c (Prod. No. BML-UW9070), G: UbcH6 (Prod. No. BML-UW8710), H: UbcH7 (Prod. No. BML-UW9080), I: UbcH8 (Prod. No. BML-UW9135), J: UbcH10 (Prod. No. BML-UW0960), K: Ubc13/MMS2 (Prod. No. BML-UW9565) respectively, using Streptavidin-HRP detection system as described in “Analysis by Western Blotting” section. M: Biotinylated SDS molecular weight markers (Sigma, SDS-6B) from bottom: 20.1, 29.0, 39.8, 58.1kDa.
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Product Specification

Application Notes:Uses:
1. Ubiquitinylation of target proteins in presence of dedicated E3 ligase. Panel of E2s provided for generation of E2-Ub thioester conjugates for testing vs. specific E3/target combinations. For example: ubiquitinylation of p53 in the presence of mdm2 (E3) and UbcH5b (E2)10.
2. Activation of Ub for thioester conjugation to novel E2 enzymes (substituted like for like with kit E2s, under directly comparable conditions).
3. Use of cell lysate or crude fractions/preparations as source of E3 ligases to facilitate ubiquitinylation of purified target proteins in the presence of ubiquitinylation kit components.
4. Substrate (target) independent in vitro ubiquitinylation reactions. Determine ubiquitin ligase activity/specificity of proposed E3 enzymes and/or their catalytic domains/fragments11.

Note: Protocols provided for applications 1 and 2. Assay set-up can be readily modified for alternative applications by inclusion, omission or substitution of specific enzyme components.

Quantity:Provides sufficient material for 4 reactions with each included E2 enzyme.
Use/Stability:All kit components should be stored at -80°C to ensure stability and activity.
Handling:Avoid freeze/thaw cycles.
Shipping:Shipped on Dry Ice
Long Term Storage:-80°C
Kit/Set Contains:

20X Ubiquitin Activating Enzyme Solution
Human recombinant E1 (His6-tagged), 125µl provided, Product No. BML-UW9410

10X Ubiquitin Conjugating Enzyme Solutions (E2)
20µl of each E2 provided
UbcH1 (His6-tagged), Prod. No. BML-UW9020
UbcH2 (His6-tagged), Prod. No. BML-UW9025
UbcH3 (His6-tagged), Prod. No. BML-UW8730
UbcH5a (His6-tagged), Prod. No. BML-UW9050
UbcH5b (His6-tagged), Prod. No. BML-UW9060
UbcH5c (His6-tagged), Prod. No. BML-UW9070
UbcH6 (His6-tagged), Prod. No. BML-UW8710
UbcH7 (His6-tagged), Prod. No. BML-UW9080
UbcH8 (His6-tagged), Prod. No. BML-UW9135
UbcH10 (untagged), Prod. No. BML-UW0960
UbcH13/Mms2 (His6-tagged), Prod. No.BML-UW9565

20X Biotinylated Ubiquitin Solution (Bt-Ub), 125µl provided, Prod. No. BML-UW8705
20X Mg-ATP Solution, 125µl provided, Prod. No. BML-EW9805
2X Non-reducing Gel Loading Buffer, 2.5 ml, Prod. No. BML-KW9880
10X Ubiquitinylation Buffer, 250 µl, Prod. No. BML-KW9885


Product Literature References

Hepatocyte TRAF3 promotes liver steatosis and systemic insulin resistance through targeting TAK1-dependent signalling: P.X. Wang, et al.; Nat. Commun. 7, 10592 (2016), Application(s): In vitro ubiquitination assays, Abstract; Full Text
Host cell-catalyzed S-palmitoylation mediates Golgi targeting of the Legionella ubiquitin ligase GobX: Y.H. Lin, et al.; J. Biol. Chem. 290, 25766 (2015), Application(s): Western Blot, Abstract; Full Text
TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING: Y. Wang, et al.; PLoS Pathog. 11, e1005012 (2015), Application(s): In vitro ubiquitination assay, Abstract; Full Text
TRIM35 negatively regulates TLR7- and TLR9-mediated type I interferon production by targeting IRF7: Y. Wang, et al.; FEBS Lett. 589, 1322 (2015), Application(s): In vitro ubiquitination assay, Abstract;
Parkin loss of function contributes to RTP801 elevation and neurodegeneration in Parkinson's disease: J. Romaní-Aumedes, et al.; Cell Death Dis. 5, e1364 (2014), Abstract;
Rlim, an E3 ubiquitin ligase, influences the stability of Stathmin protein in human osteosarcoma cells: X. Chen, et al.; Cell Signal. 26, 1532 (2014), Abstract;
Stability of the human pregnane X receptor is regulated by E3 ligase UBR5 and serine/threonine kinase DYRK2: S.S. Ong, et al.; Biochem. J. 459, 193 (2014), Abstract;
Ubiquitin-proteasome-mediated degradation of S-RNase in a solanaceous cross-compatibility reaction: T. Entani, et al.; Plant J. 78, 1014 (2014), Abstract;
E3 ubiquitin ligase RNF126 promotes cancer cell proliferation by targeting the tumor suppressor p21 for ubiquitin-mediated degradation: X. Zhi, et al.; Cancer Res. 73, 385 (2013), Abstract;
The E3 ubiquitin ligase MARCH8 negatively regulates IL-1β-induced NF-κB activation by targeting the IL1RAP coreceptor for ubiquitination and degradation: R. Chen, et al.; Proc. Natl. Acad. Sci. U.S.A. 109, 14128 (2012), Abstract; Full Text
TRIM32 protein modulates type I interferon induction and cellular antiviral response by targeting MITA/STING protein for K63-linked ubiquitination: J. Zhang, et al.; J. Biol. Chem. 287, 28646 (2012), Abstract;
Cullin 4B protein ubiquitin ligase targets peroxiredoxin III for degradation: X. Li, et al.; J. Biol. Chem. 286, 32344 (2011), Abstract; Full Text
Tripartite motif 8 (TRIM8) modulates TNFα- and IL-1β-triggered NF-κB activation by targeting TAK1 for K63-linked polyubiquitination: Q. Li, et al.; Proc. Natl. Acad. Sci. U.S.A. 108, 19341 (2011), Abstract; Full Text
1-Benzyl-1,2,3,4-tetrahydroisoquinoline binds with tubulin beta, a substrate of parkin, and reduces its polyubiquitination: R. Kohta, et al.; J. Neurochem. 114, 1291 (2010), Abstract;
Pasteurella multocida Toxin-induced Pim-1 expression disrupts suppressor of cytokine signalling (SOCS)-1 activity: D. Hildebrand, et al.; Cell. Microbiol. 12, 1732 (2010), Abstract;
The E3 ubiquitin ligase RNF5 targets virus-induced signaling adaptor for ubiquitination and degradation: B. Zhong, et al.; J. Immunol. 184, 6249 (2010), Abstract; Full Text
The Fbw7 tumor suppressor targets KLF5 for ubiquitin-mediated degradation and suppresses breast cell proliferation: D. Zhao, et al.; Cancer Res. 70, 4728 (2010), Abstract; Full Text
REUL is a novel E3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-I: D. Gao, et al.; PLoS One 4, e5760 (2009), Abstract; Full Text

General Literature References

Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo: M.K. Saville, et al.; J. Biol. Chem. 279, 42169 (42169), Abstract;
Characterization of the novel E3 ubiquitin ligase encoded in exon 3 of herpes simplex virus-1-infected cell protein 0: R. Hagglund, et al.; PNAS 99, 7889 (2002), Abstract;
Mechanisms underlying ubiquitination: C.M. Pickart, et al.; Annu. Rev. Biochem. 70, 503 (2001), Abstract;
Functions of the MDM2 oncoprotein: D.A. Freedman; Cell Mol. Life. Sci. 55, 96 (1999), Abstract;
Regulation of the p53 tumor suppressor protein: M. Oren, et al.; J. Biol. Chem. 274, 36031 (1999), Abstract;
The p53 pathway: C. Prives, et al.; J. Pathol. 187, 112 (1999), Abstract;
The ubiquitin-proteasome system and endocytosis: G.J. Strous, et al.; J. Cell. Sci. 112 , 1417 (1999), Abstract;
The ubiquitin system: A. Hershko, et al.; Annu. Rev. Biochem. 67, 425 (1998), Abstract;
Pathways of ubiquitin conjugation: A.L. Haas, et al.; FASEB J. 11, 1257 (1997), Abstract;
P53 and mdm-2: interactions between tumor suppressor gene and oncogene products: M. Perry, et al.; Mt. Sinai J. Med. 61, 291 (1994), Abstract;
The p53 tumor suppressor gene: A.J. Levine, et al.; J. Lab. Clin. Med. 123, 817 (1994), Abstract;

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