Nature research paper: USP14-regulated allostery of the human proteasome by time-resolved cryo-EM
and 5 mM ATP) at 37 °C. Purified recombinant USP14 variants were incubated with proteasome for 20 min at room temperature before initiating the degradation reaction. The reaction mixtures were incubated at 37 °C for 0, 0.5, 1.0 and 2.0 min, or 10 °C for 0, 0.5, 1.0, 5.
After 30 min of room temperature incubation, the absorbance at 620 nm was determined using a Varioskan Flash spectral scanning multimode reader .Peptide hydrolysis by the human proteasomes was measured using fluorogenic substrate Suc-LLVY-AMC . Human proteasomes were incubated with USP14 variants in buffer , 100 mM KCl, 0.5 mM MgClBSA) for 20 min at room temperature. 10 μM Suc-LLVY-AMC was added to the reaction mixture, which was incubated for 30 min at 37 °C in the dark.
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