Yarra sec 2000 column

HPLC–SEC analyses and purifications were performed using a Phenomenex (Bologna, Italy) Yarra SEC-2000 column (300 × 7.8 mm, 3 µm) eluted with 90 mM KCl and 10 mM KH₂PO₄/CH₃CN (80:20, v/v), flow rate 0.6 mL/min, UV-detector at 260 nm. The analyses were performed at room temperature.

Gel filtration chromatography (GFC) was performed on a Knauer HPLC system consisting of a K-1001 pump, S 2600 photo diode array detector, and S 3800 autosampler (Knauer, Berlin, Germany). Samples (50 μL) were injected onto a Yarra SEC-2000 column (300 mm × 7.8 mm i.d, 3 μm; Phenomenex, Torrance, CA, USA), and were eluted at 1 mL/min with 0.05 M phosphate buffer containing 0.3 M sodium chloride, pH 6.9. Column eluent was monitored at 214, 230, 260 and 280 nm. Data were acquired and analysed using Chromgate 3.1 software (Knauer).

The extracts obtained after SBW treatment of codfish frames were filtrated through nylon syringe filters (0.22 µm pore diameter, ALWSCI Corporation, Zhejiang, China) and submitted to size exclusion–gel permeation chromatography (SEC-GPC) to estimate their molecular mass distribution. The equipment used was a Knauer Smartline HPLC system. For each sample, 20 µL were injected into a Phenomenex Yarra SEC-2000 column (300 × 7.8 mm i.d.) maintained at a constant temperature of 25 °C, and eluted with 0.1 M phosphate buffer (pH 6.8) including 0.025% sodium azide at a flow rate of 1.0 mL/min. The relative intensity of the eluate was monitored with a UV/Vis detector at 280 nm. The retention times of molecular mass standards (Protein Standard Mix 69385 from Sigma-Aldrich (Saint Louis, MO, USA)) were used to calculate the molecular mass distribution of the SBW extracts according to the retention time of the different peaks present in the chromatogram.

The HeLa lysate was centrifuged (15,000 rpm at 4 °C for 15 min), and the clarified supernatant was loaded onto HisPur™ Ni-NTA Spin Columns (Pierce Biotechnology, Rockford, IL, USA), followed by the addition of the binding and elution buffers (50 mM sodium phosphate and 300 mM sodium chloride (PBS) without 10 mM imidazole at pH 7.2 and 5.8, respectively). The purified protein was then analyzed by UPLC and SEC on an HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) using a Yarra SEC-2000 column (300 × 7.8 mm; Phenomenex, Torrance, CA, USA). For chromatographic separation, solvent A (150 mM NaCl and 50 mM sodium in 50 mM sodium phosphate; pH 6.5) was equilibrated with a linear gradient of solvent B (water with 0.1% TFA, 99.5% purity; Sigma-Aldrich, St. Louis, MO, USA) over 70 min at a flow rate of 1 mL min⁻¹. The purity (>95.0%) of the N-terminally labeled protein was confirmed by analytical SEC-HPLC (Agilent Technologies, Inc., Santa Clara, CA, USA). Western blotting was per formed using a Simple Western™ system (WES, ProteinSimple, San Jose, CA, USA) and an anti-penta-His antibody (1:1000 in TBST; Qiagen, Hilden, Germany) for detection (data not shown).

Purity and Stokes radii of the recombinant fusion proteins were determined by HPLC size exclusion chromatography with a Yarra SEC–2000 column (Phenomenex, Aschaffenburg, Germany). 20 μl of proteins (0.5 mg/ml) were injected at a flow rate of 0.5 ml/min. The following standard proteins were used to calculate the apparent molecular mass and hydrodynamic radius: thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), BSA (67 kDa), carbonic anhydrase (29 kDa) and FLAG peptide (1 kDa).

HPLC-SEC was performed using a Phenomenex (Bologna, Italy) Yarra SEC-2000 column (300 × 7.8 mm, 3 µm) eluted with 90 mM KCl and 10 mM KH₂PO₄/CH₃CN (80:20, v/v), flow rate 0.6 mL/min, UV-detector at 260 nm. The analyses were performed at room temperature.