Product Name :
Ubiquitinylation kit
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All kit components should be stored at -80°C to ensure stability and activity.
Description:
Versatile tool for generation of ubiquitin-E2 thioesters. 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 , B: UbcH2 , C: UbcH3 , D: UbcH5a , E: UbcH5b , F: UbcH5c , G: UbcH6 , H: UbcH7 , I: UbcH8 , J: UbcH10 , K: Ubc13/MMS2 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. 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 , B: UbcH2 , C: UbcH3 , D: UbcH5a , E: UbcH5b , F: UbcH5c , G: UbcH6 , H: UbcH7 , I: UbcH8 , J: UbcH10 , K: Ubc13/MMS2 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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| Application Notes Uses:1. Ubiquitinylation of target proteins in presence of dedicated E3 ligase.356547-88-1 medchemexpress Panel of E2s provided for generation of E2-Ub thioester conjugates for testing vs.1448347-49-6 site specific E3/target combinations.PMID:30247825 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. | 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 | Quantity Provides sufficient material for 4 reactions with each included E2 enzyme.
