Acquity ultra performance system

Polyphenol composition was obtained through ultra-high-performance liquid chromatography (UPLC) using an ACQUITY Ultra Performance system linked to a PDA 2996 photodiode array detector (Waters, Milford, MA, USA), linked to an Empower software (Waters). The analysis was performed following the method of Ombra et al. [17 (link)] at λ = 280 nm with a reversed-phase column (BEH C₁₈, 1.7 µm, 2.1 mm× 100 mm, Waters), at 30 °C, at a flow rate of 250 μL/min, and with pressure ranging from 6000 to 8000 psi. The effluent was introduced to an LC detector (scanning range 210–400 nm, resolution 1.2 nm). The injection volume was 5 μL. Phenolic compounds were identified and quantified through comparison of the peak areas on the chromatograms of samples with those of diluted standard solutions.

For the pulldown with full-length RILPL2, nucleotide exchange was performed using purified WT Rab8a incubated in 10 mM EDTA for 10 minutes at room temperature in the presence of 10X molar excess GDP. The exchange was terminated by addition of 15 mM MgCl₂ and excess nucleotides were removed by running samples through a PD10 column (GE healthcare), or by immediate gel filtration chromatography. To verify successful exchange, 100 μL the protein (>1mg/mL) was boiled for 10 min at 95°C to denature the protein and release the nucleotide, followed by centrifugation for 30 min 16,000 x g, 4°C to remove precipitated protein. The supernatant was mixed with running buffer (100 mM potassium phosphate, 8 mM thiobarbituric acid, pH 6.5) at a 1:1 ratio. The samples were loaded on an Acquity Ultra Performance system (Waters Corporation, Milford, MA, USA; or Varian 920 LC machine, Agilent, Stockport, UK) equipped with a ZORBAX 300SB-C18 column (Agilent, Stockport, UK). Elution profiles of GMP, GDP, GTP (Sigma Aldrich) and GppNHp (Jena Bioscience, Germany) were subjected to HPLC and compared with Rab8a. The nucleotide state of Rab8a(Q67L) was confirmed to be GTP-bound using the analytical HPLC strategy.