Synergi c18 column 150 4.6mm

SAMHD1 dNTPase activity assay was performed with 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 0.5 mM TCEP and 1 mM substrate dGTP in a 500-μl reaction volume. Reactions were initiated at 37 °C by the addition of purified SAMHD1 samples to obtain a final concentration of 500 nM. Aliquots of reactions were collected at various times points and terminated by fivefold dilution into ice-cold buffer containing 10 mM EDTA, which was followed by spinning through an Amicon Ultra 0.5 ml 10-kDa filter (Millipore) at 16,000g for 20 min. Deproteinized samples were analyzed by HPLC using a Synergi C18 Column 150 × 4.6 mm (Phenomenex), pre-equilibrated in 20 mM ammonium acetate, pH 4.5 (buffer A). Samples were eluted with a methanol (buffer B) gradient over 14 min at a flow rate of 1 ml/min, and the elution profiles (UV absorption at 260 nm) were recorded.

All SAMHD1 dNTPase activity assays were performed in a reaction buffer (100 μL per reaction) containing 50 mM Tris-HCl, pH 7.5, 150 Mm NaCl, 5 mM MgCl₂ and 0.5 mM TCEP. Recombinant SAMHD1 was phosphorylated by BGLF4 or CDK1 in vitro. Non-phosphorylated SAMHD1 was similarly processed by incubating with kinase-dead BGLF4 (BGLF4-KD) or Buffer control. Then phosphorylated and non-phosphorylated SAMHD1 (1 μM) were converted to GTP-bound dimer by adding 1mM GTP for 1 min. SAMHD1 dimer was further activated by adding dNTP mixture (12.5 μM each) for 1 min. The mixture was diluted 10-fold and the substrate dCTP, dTTP, dATP or dGTP (100 μM) is added for 0 to 240 min at room temperature. The reaction samples were collected at 0, 120 and 240 min and quenched by 10-fold dilution into ice cold buffer containing 10 mM EDTA, followed by spinning through an Amicon Ultra 0.5 mL 10 kDa Alter at 16000 xg for 20 min (Jang et al., 2016 (link); Tang et al., 2015 (link)).
Deproteinized samples were analyzed by HPLC using a Synergi C18 Column 150 × 4.6mm (Phenomenex). The column was pre-equilibrated in 20mM ammonium acetate, pH 4.5 (buffer A). Injected samples (100 μl) were eluted with a linear methanol gradient over 14 min at a flow rate of 1 mL/min. The dN yields were quantified by integration of the calibrated UV absorption peak at 260 nm.