Ubiquitinylation kit

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.

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 (His 6 -tagged), Prod. No. BML-UW9020 UbcH2 (His 6 -tagged), Prod. No. BML-UW9025 UbcH3 (His 6 -tagged), Prod. No. BML-UW8730 UbcH5a (His 6 -tagged), Prod. No. BML-UW9050 UbcH5b (His 6 -tagged), Prod. No. BML-UW9060 UbcH5c (His 6 -tagged), Prod. No. BML-UW9070 UbcH6 (His 6 -tagged), Prod. No. BML-UW8710 UbcH7 (His 6 -tagged), Prod. No. BML-UW9080 UbcH8 (His 6 -tagged), Prod. No. BML-UW9135 UbcH10 (untagged), Prod. No. BML-UW0960 UbcH13/Mms2 (His 6 -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