Nucleotide Quantification by Capillary Electrophoresis

Culture medium was quickly aspirated from cells grown on 6 cm culture dishes, and cells were washed with 1 ml of ice-cold PBS. After rapid aspiration of PBS, a minimal volume of ice-cold 5% perchloric acid was added and the samples vortexed to ensure complete lysis. After centrifugation (14,000 rpm, 4°C, 3 min) to remove acid-insoluble material, the supernatant was extracted with two washes of an equal volume of 1:1 tri-n-octylamine and 1,1,2-trichlorotrifluoroethane. The nucleotides remaining in the aqueous phase were then separated by capillary electrophoresis with on-column isotachophoretic concentration, using run buffers consisting of 50 mM Na phosphate and 50 mM NaCl (pH 5.2; leading buffer) and 100 mM MES/Tris (pH 5.2; tailing buffer). To each buffer was added 0.2% hydroxyethylcellulose to decrease electro-osmotic flow. Nucleotide peaks were detected by UV absorbance at 260 nM and integrated using System Gold software (Beckman). Nucleotide ratios were calculated from peak areas after correction for retention times. Identification of peaks as ATP, ADP, and AMP were confirmed by additional runs spiked with internal standards and analysis of absorbance spectra of individual peaks.