Ni nta agarose

Ddi2 and 5 nM purified proteasomes were combined in 25 mM Hepes–KOH (pH 8), 125 mM potassium acetate, 1 mM DTT, 2.5 mM MgCl₂, 0.1 mg/ml bovine serum albumin (BSA), 0.05% Triton X-100, 1 mM ATP, and a constant volume of Ni–NTA agarose (Gold Biotechnology; catalog no.: H-350) at 4 °C for 30 min. Resin was separated from the unbound fraction by centrifugation (100g, 2 min). The column was washed three times with 10 column volumes of 50 mM Tris (pH 7.5), 10 mM MgCl₂, 40 mM KCl, 1 mM ATP, and 1 mM DTT. Proteasome activity on the resin was measured in 50 mM Tris (pH 7.5), 10 mM MgCl₂, 40 mM KCl, 1 mM ATP, 1 mM DTT, and 20 μM Suc-Leu-Leu-Val-Tyr-amc at λ_excitation 380 nm, λ_emission 460 nm, and 37 °C for 30 min. Captured proteasome activity is reported directly as arbitrary fluorescence units.