System gold 166 detector

To measure dNTP triphosphohydrolase activity of SAMHD1, 1.6 μM recombinant SAMHD1-GST (SAMHD1) was incubated with different 500 μM nucleoside-5'-triphosphate substrates in the presence of 500 μM dCMP, 500 μM GTP and reaction buffer (50 mM Tris-HCl [pH 8], 100 mM KCl, 5 mM MgCl₂, and 0.1% Triton X-100). Reactions were incubated for 2 h at 37°C and terminated by incubation for 10 min at 75°C. Reactions were separated and quantified by anion exchange HPLC method [32 (link)]. Separation was done using two DNAPac PA100 columns equilibrated with running buffer (25 mM Tris–HCl [pH 8] and 0.5% acetonitrile) for 10 min, 30 μL sample was injected and eluted with a linear gradient of 240 mM NH₄Cl for 12 min, run at an isocratic gradient with 240 mM NH₄Cl for 5 min, and column was again equilibrated with running buffer (Beckman Coulter System Gold 126 Solvent Module). Absorbance was measured with a Beckman Coulter System Gold 166 Detector at 254 nm. The amounts of deoxycytidine-5'-monophosphate (dCMP), dGTP and (deoxy)nucleoside-5'-TP analogs were determined by integrating the peak area using 32 Karat 8.0 Software. Data was normalized to dCMP peak area for each sample, used as a sample loading control. Determining changes for different (deoxy)nucleoside-5'-triphosphates of interest was calculated by setting sample without SAMHD1 peak area to 100%.

Free amino acids were measured in all biopsies collected. Amino acids were determined using HPLC according to the Pico-Tag method of Water as reported earlier (Caputo et al. 1998b (link)). In brief, frozen biopsy specimens were crushed in liquid nitrogen and the resultant powder was extracted with 4.8% perchloric acid. Neutralized 100 μL of the extract was dried immediately after extraction using vacuum centrifugation (Savant SV160). Free amino acids were derivatized by phenylisothiocyanate (PITC) and were separated by 30 cm Pico-Tag column (Millipore Corporation, USA) with two Beckman delivery pumps at a constant flow rate of 1 ml/min with gradient elution for 25 min at 46°C. Derivatized amino acids were detected at 254 nm using a Beckman System Gold 166 Detector. Quantitative analysis was carried out using amino acid standards (Thermo, UK) and the acquired data were processed using 32 Karat software supplied by Beckman. Concentration of metabolites was expressed as nmol/mg wet weight.