Gf c glass microfiber filter

To demonstrate the reaction mechanism between the mica minerals and the collector (Armac-T) used in this study, Fourier transform infrared spectrometry (FT-IR, IRTracer-100, Shimadzu, Kyoto, Japan) was used. The Armac-T (10,000 mg/kg-sample) was injected into 1 g of each pure mineral (i.e., muscovite, biotite, and quartz) and reacted for 10 min at pH 3.5 ± 0.2. Then, the sample was filtered using a Whatman GF/C glass microfiber filter (1.2 μm pore size), dried at room temperature, and analyzed using the FT-IR with the aid of attenuated total reflectance to avoid further pretreatment. The transmittance was measured at a resolution of 4 cm⁻¹ in a range of 4000–500 cm⁻¹.

In vitro aminoacylation assay was conducted according to the previous papers^{67 (link),68 (link)}. For chimeric Phe and Ala system, 100 nM synthetases were incubated with 0, 30 μg of extracted tRNAs in reaction buffer (50 mM Tris–HCl, 10 mM MgCl₂, 20 mM KCl, 2 mM DTT, pH 7.4) supplemented with 4 mM ATP, 0.1 mg/ml BSA and 80 μM L-phenylalanine, 17.8 μM L-[¹⁴C]-phenylalanine for chimeric Phe system or 50 μM L-alanine, 33.3 μM L-[¹⁴C]-alanine for chimeric Ala system at 37 °C for 40 min. After incubation, the aliquots were spotted onto Whatman GF/C glass microfiber filter pre-equilibrated with 10% cold TCA. The paper filters were washed with cold 10% TCA four times and then 95% ethanol four times. After washing, the filter papers were visualised by the liquid scintillation counter to quantify the radioactivity. For chimeric His system, 25 nM synthetase was incubated with 0, 10 and 30 μg of extracted tRNAs in reaction buffer (50 mM Tris–HCl, 10 mM MgCl₂, 20 mM KCl, 2 mM DTT, pH 7.4) supplemented with 0.1 mM ATP, 1 mM L-histidine and 0.1 mg/ml BSA at 37 °C for 2 h. The aminoacylation was measured through the production of AMP with AMP-Glo assay (Promega) according to the manufacturer’s instructions.

PARP2 (30 nM final) was pre-incubated with varying concentrations of p18mer DNA (0.01–200 nM final; sequence of p18mer: top strand 5’P-GGGTTGCGGCCGCTTGGG-3’OH, bottom strand 5’-CCCAAGCGGCCGCAACCC-3’OH, purchased as a duplex from Integrated DNA Technologies) in a buffer containing 50 mM Tris pH = 8, 50 mM NaCl, 1 mM MgCl₂, 0.1 mM EDTA and 0.5 mg/mL bovine serum albumin in 96-well plates (Costar 3898). Following addition of ³²P-NAD⁺ (40–80 μM, 1.8 x 10⁶ cpm/well) to yield a final volume of 25 μL, reactions were quenched after 30 s by addition of 50 μL of 30% trichloracetic acid (TCA). Samples (50 μL of total) were then loaded onto a Whatman Mini-Fold Spot-Blot apparatus containing a Whatman GF/C glass microfiber filter. Each well was washed three times with 100 μL of 10% TCA. After removal of the filter from the apparatus, the filter was gently incubated in 20–40 mL of 10% TCA for three more washes. After drying, the filter was exposed to a phosphoimager screen (GE Healthcare) and imaged using a Typhoon 9500. Spot intensities were quantitated using ImageQuant.