Rapid Kinetic Analysis of Ubiquitin Transfer

Reactions were assembled in two separate mixtures: an E1/E2 mix that contained excess unlabeled peptide (tube 1) and an SCF-³²P-labeled substrate mix (tube 2; see Supplementary file 2 for concentrations). Following addition of E2 and/or ARIH1 to tube 1 already containing reaction buffer (30 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl₂, 2 mM DTT and 2 mM ATP) and ubiquitin, each mix was incubated for at least 8 min while being loaded into separate sample loops on a KinTek RQF-3 quench flow instrument. Reactions were initiated by bringing the two mixes together in drive buffer (30 mM Tris-HCl (pH 7.5), 100 mM NaCl), and then quenched at various time points in reducing 2x SDS-PAGE loading buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 30 mM EDTA, 4% SDS, and 4% beta-mercaptoethanol). Each reaction was performed at least in duplicate, and time points were resolved on 18% polyacrylamide SDS-PAGE gels. Autoradiography was performed using a Typhoon 9410 Imager and Image Quant software (GE Healthcare). Each product species was quantified as a fraction of the total signal of its respective lane. The rates of ubiquitin transfer were determined by fitting to analytical closed-form solutions (Pierce et al., 2009 (link)) using Mathematica.