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Ubiquitinylation kit

Versatile tool for generation of ubiquitin-E2 thioesters.
BML-UW9920-0001 50 tests 464.00 USD
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-KW9020), B: UbcH2 (Prod. No. BML-KW9025), C: UbcH3 (Prod. No. BML-KW9030), D: UbcH5a (Prod. No. BML-KW9050), E: UbcH5b (Prod. No. BML-KW9060), F: UbcH5c (Prod. No. BML-KW9070), G: UbcH6 (Prod. No. BML-KW8710), H: UbcH7 (Prod. No. BML-KW9080), I: UbcH8 (Prod. No. BML-KW9135), J: UbcH10 (Prod. No. BML-KW8715), K: Ubc13/MMS2 (Prod. No. BML-KW9565) 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: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 50 x 50μl reactions.
Use/Stability:All kit components should be stored at -80°C to ensure stability and activity.
Handling:Avoid freeze/thaw cycles.
Long Term Storage:-80°C
Kit/Set Contains:

20X Ubiquitin Activating Enzyme Solution (Human recombinant E1 (His6-tagged)
125μL provided
10X Ubiquitin Conjugating Enzyme Solutions (E2)  
20μL of each E2 provided
UbcH1 (His6-tagged)
UbcH2 (His6-tagged)
UbcH3 (His6-tagged)
UbcH5a (His6-tagged)
UbcH5b (His6-tagged)
UbcH5c (His6-tagged) 
UbcH6 (His6-tagged)
UbcH7 (His6-tagged) 
UbcH8 (His6-tagged)
UbcH10 (His6-tagged) 
UbcH13/Mms2 (His6-tagged)
20X Biotinylated Ubiquitin Solution (Bt-Ub) 125μL provided
20X Mg-ATP Solution 125μL provided
2X Non-reducing Gel Loading Buffer 2.5 ml
10X Ubiquitinylation Buffer 250 µl


Product Literature References

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
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 A.J. Levine et al.; J. Lab. Clin. Med. 123, 817 (817), Abstract;
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;
A. Silver and K.J. Thorn; Lab. Clin. Med. 123, 817 (1994),
P53 and mdm-2: interactions between tumor suppressor gene and oncogene products: M. Perry et al.; Mt. Sinai J. Med. 61, 291 (1994), Abstract;

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