Lc 10ai hplc system

Fractionation of crude histones was performed according to a procedure described by Shechter et al.21 (link) with a slight modification. In brief, crude histones were dialyzed against Milli-Q water, and then the protein concentrations were determined using a BCA Protein Assay Kit following the manufacturer’s instructions. We separated crude histones (50 μg) using Aeris Widepore 3.6 μm XB-C8 column (Phenomenex) fitted to an LC-10Ai HPLC system (Shimadzu, Kyoto, Japan), that equilibrated with buffer A (5% acetonitrile, 0.1% trifluoroacetic acid (TFA)) and eluted with a linear gradient of 0%–0%–35%–35%–50% buffer B (90% acetonitrile, 0.1% TFA) over 0–5–15–25–62.5 min at a flow rate of 0.5 ml/min. We monitored elution by UV absorbance at 214 nm and subjected fractions to WB.

An RhrER 2718 sample was incubated for 10 min at 99 °C to release the cofactor; denatured protein was removed by centrifugation at 14000 rpm for 30 min. The supernatant was spun through a Microcon YM3 (MWCO: 3000 Da, Merck Millipore) centrifugal concentrator device (14,000 rpm, 30 min) to remove residual protein. The resulting sample was analysed by high-performance liquid chromatography (HPLC) to identify the flavin cofactor. For the separation and quantification of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), a reverse phase C18 HPLC column connected to a Shimadzu LC10Ai HPLC system was used (Shimadzu Benelux, ‘s-Hertogenbosch, The Netherlands). Ammonium acetate (50 mM, pH 6.0) and 70% acetonitrile in ammonium acetate (50 mM, pH 6.0) were used as mobile phase. The retention times of FAD and FMN are 6.53 and 9.12 min, respectively (Supporting Information, section 4).

The acid‐quenched sample solutions obtained from the above experiments were thawed and analyzed with a Shimadzu LC‐10Ai HPLC system equipped with a 1 mL sample solution loop and an analytical RP column (TSKgel ODS‐100V 4.6 × 150 mm; Tosoh, Tokyo, Japan), which was equilibrated at 25 °C with 59 : 41 (v/v) mixture of 0.1% TFA in H₂O (eluent A) and 0.1% TFA in acetonitrile (eluent B) at a flow rate of 0.5 mL·min⁻¹. After injection of the sample solution (1 mL), a gradient of acetonitrile (the ratio of eluent B linearly increased from 41% to 45% in 0–40 min and from 45% to 100% in 40–42 min) was applied. The folding intermediates were detected by UV (SPD‐M10A vp; Shimadzu, Kyoto, Japan) at 280 nm. The recorded signals were integrated and analyzed by using lc solution software (Shimadzu).