Online Purchasing Account You are logged on as Guest. LoginRegister a New AccountShopping cart (Empty)
United States 

Ubiquitinylation kit

Versatile tool for generation of ubiquitin-E2 thioesters.
 
BML-UW9920-0001 1 Kit 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.
bml-uw9920
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.
Please mouse over
bml-uw9920

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.
 
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

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

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10065155: 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;

Related Literature

Product Flyers
Integrated Solutions for Stem Cell Discovery
Integrated Solutions for Stem Cell Discovery 05-Jun-13
Download as PDF

Catalogs
Ubiquitin & UBL Signaling Catalog
Ubiquitin & UBL Signaling Catalog 02-Oct-09
Download as PDF

All new literature pieces

Recommend this page

 
Keep in touch

©2014 Enzo Life Sciences, Inc.,