Ubiquitylation assays

1) Make the E3 mixes. For these assays, it would contain UHRF1, HeDNA, H3 peptide, and whatever else you want in your assay. This E3 mix will get added 1:1 with the ubiquitin mix to initiate the reaction. I typically use 20μL reactions, but any volume you want should be fine as long as you add 1:1 with the ubiquitin mix or adjust your ubiquitin mix concentration. 

2) Make the ubiquitin mix. This tube should contain DTT (1mM) MgCl (2.5-5mM) Ubiquitin (8mM-100mM) ATP (10mM) E2-Ube2D (200nM-2μM) E1 (50nM-100nM) NaCl 100mM. The concentration in parentheses are the final concentration when mixing in the final reaction, so in the ubiquitin mix these concentrations are 2-fold higher. I recommend making one mix big enough for all your assays. Be sure to mix this well and do not keep this tube or the E3 tubes on ice or the reaction will start slower. 

3) Initiate the reaction by mixing the ubiquitin mix with the E3 mix. I usually set up the assay so it is a 1:1 mix (i.e. add 10μL of ubiquitin mix to 10μL of E3 mix). Let run at room temperature for 10-20 minutes.

4) Quench the reaction with SDS-loading dye and run a gel. In this paper, we used FLAG-ubiquitin and detected ubiquitin via FLAG western blot, but there are other ways to visualize ubiquitin (Flourescent ubiquitin, radiolabeled ubiquitin, anti-ubiquitin antibody, fluorescent substrate). There are other ways to quench the reaction also, (using EDTA or DTT also works). 

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