Agilent 1000 series hplc

The oligomeric state of His-CleD-GFP in the presence or absence of c-di-GMP was determined using a S200 5/150 column (GE Healthcare) mounted to an Agilent 1000 series HPLC (Agilent Technologies). The setup was connected with a miniDwan TriStar multiangle light scattering detector (Wyatt Technology) and Optilab rRex refractive index detector (Wyatt Technology). CleD-GFP (20 µl) was loaded on the column in the absence or presence of c-di-GMP at 5:1 molar ligand/protein ratio. Flow rate was 0.5 ml/min using SEC buffer (see protein expression and purification in the main section). Experiments were performed at 6°C. The mass distribution and molecular weight of samples were determined using the ASTRA five software (Wyatt Technology). The extinction coefficient of c-di-GMP were ε₂₅₃ = 28,600 M-1 cm⁻¹ and ε₂₈₀ = 17160 M⁻¹ cm⁻¹ and for CleD-GFP, an ε₂₈₀ = 31860 M⁻¹ cm⁻¹ was calculated using EXPASY server.

Monosaccharide derivatives were analyzed in Agilent 1000 series HPLC (Agilent Technologies, Model No.1100). A reversed-phase C₁₈ column (ZORBAX 300 SB-C18, 5 μm, 4.6 × 250 mm, USA) and 1-butylamine-phosphoric acid-tetrahydrofuran mobile phase system consisting of solvents A and B were used for this analysis. Solvent A comprises of 0.2% 1-butylamine, 0.5% phosphoric acid, and 1% tetrahydrofuran in water, and solvent B consisted of equal parts of solvent A and acetonitrile. The separations were carried out at 24 °C using a flow rate of 1 mL/min, and 20 mL of each sample was injected. An UV detector was used to detect the derivatized monosaccharides. The gradient program was used according to [21 (link)].