Drp1 nucleotide state

To determine the nucleotide state of purified Drp1, samples were prepared of 150 μM GTP, 75 μM GDP, and 160 μM Drp1. Samples were diluted into 20 mM HEPES, pH 7.5, 150 mM KCl, 2 mM MgCl₂, 1 mM DTT, and 0.5 mM EDTA and then boiled for 10 min and centrifuged for 10 min at 13,000 rpm in a microcentrifuge to denature and remove protein. The supernatant was diluted 1:5 with 10 mM Tris, pH 8.0, and 1 mM MgCl₂. We loaded 400 μl of sample onto a 1-ml SourceQ15 column (GE Biosciences). A linear gradient from 0 to 150 mM was used to elute nucleotides. Absorbance at 253 nm was used to detect nucleotide.