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 Details
| 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. |
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| Quantity: | Provides sufficient material for 4 reactions with each included E2 enzyme.
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| Use/Stability: | All kit components should be stored at -80°C to ensure stability and activity. |
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| Handling: | Avoid freeze/thaw cycles. |
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| Shipping: | Shipped on Dry Ice |
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| Long Term Storage: | -80°C |
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| Contents: | 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 |
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| Regulatory Status: | RUO - Research Use Only |
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Product Literature References
RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork: B. Kavli, et al.; Nucleic Acids Res. 49, 3948 (2021), Abstract;
The RING-type protein BOI negatively regulates the protein level of a CC-NBS-LRR in Arabidopsis: J. Huang, et al.; Biochem. Biophys. Res. Commun. 578, 104 (2021), Abstract;
The RNA-binding protein HuR is a novel target of Pirh2 E3 ubiquitin ligase: A. Daks, et al.; Cell Death Dis. 12, 581 (2021), Abstract; Full Text
Crystal structure of GCN5 PCAF N-terminal domain reveals atypical ubiquitin ligase structure: S. Toma-Fukai, et al.; J. Biol. Chem. 295, 14360 (2020), Abstract;
Ehrlichia chaffeensis TRP120-mediated ubiquitination and proteasomal degradation of tumor suppressor FBW7 increases oncoprotein stability and promotes infection: J.Y. Wang, et al.; PLoS Pathog. 16, e1008541 (2020), Abstract; Full Text
Enhanced sustainable green revolution yield via nitrogen-responsive chromatin modulation in rice: K. Wu, et al.; Science 367, eaaz2046 (2020), Abstract;
RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells: K.M. Tullett, et al.; Elife 9, e63452 (2020), Abstract; Full Text
TRIB3 supports breast cancer stemness by suppressing FOXO1 degradation and enhancing SOX2 transcription: J.M. Yu, et al.; Nat. Commun. 10, 5720 (2019), Abstract; Full Text
A Lotus japonicus E3 ligase interacts with the Nod Factor Receptor 5 and positively regulates nodulation: D. Tsikou, et al.; BMC Plant Biol. 18, 217 (2018), Abstract; Full Text
Autoubiquitination of feline E3 ubiquitin ligase BCA2: W. Wang, et al.; Gene 638, 1 (2018), Abstract;
The deubiquitinating enzyme cylindromatosis mitigates nonalcoholic steatohepatitis: Y.X. Ji, et al.; Nat. Med. 24, 213 (2018), Abstract;
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