Amicon ultra 0.5 ml 10 kda filter

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.

In vitro cA₄ cleavage assays were performed by incubating 2 μM Card1 or Csm6 with 100 μM cA4 in 150 μL reaction buffer containing 20 mM Tris–HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, at 37°C (for Card1) or 55°C (for Csm6) in a time course. At desired time points, the protein was separated by using an Amicon Ultra 0.5-ml 10-kDa filter (Millipore) at 10,000 × g for 20 min at 4°C. The filtered 100 μL reactions were analyzed using a 1 mL monoQ column (GE Healthcare) on an ÄKTA Pure system (GE Healthcare). Elution was performed with a linear gradient from 0.1 M to 1 M NaCl at a flow rate of 0.5 ml/min. The UV absorbance data recorded at 260 nm were employed, and final graphs were represented by GraphPad Prism v8.0.