Kta purifier hplc system

Hundred microliters of each FP were prepared in 0.5 mg/ml and applied to Superdex 200 Increase 10/300 GL columns (GE healthcare, Chicago, IL, USA) using PBS (20 mM, pH 7.4) at a flow rate of 0.8 ml/min. Protein elution was monitored by measuring optical absorbance at 280 nm (using an ÄKTA Purifier HPLC system, GE Healthcare). To determine the apparent molecular weight, Gel Filtration Calibration Kit LMW (GE Healthcare) was used.

AIF_Δ101 was incubated with its different nuclear protein partners (1:3 ratio) for 15 min at 25°C in 50 mM potassium phosphate, pH 7.4. Samples were then loaded into a Sephadex S-200 High Resolution (GE Healthcare) column connected to an Äkta Purifier HPLC system (GE Healthcare). Protein elution was performed in 50 mM potassium phosphate, 10 mM NaCl, pH 7.4, at a flow rate of 0.4 mL min⁻¹. The column was previously calibrated with the GE Healthcare low molecular weight calibration kit (six proteins in the 6.4 to 160 kDa range). The obtained chromatograms were fitted to a set of Gaussian functions.

Crude Daboia r. russelii venom was fractionated on Hiload^TM 16/600 Superdex 75 prep grade column (1x120 ml) pre-equilibrated with 50 mM of Tris-Cl pH 7.4 using Äkta Purifier HPLC system, GE Healthcare (Uppsala, Sweden). Fractionation was carried out at a flow rate of 1 ml/min under isocratic conditions with the same buffer. Each eluted fraction was assayed for PLA₂ and anticoagulant activity. P6 with highest anticoagulant and PLA₂ activity was further subjected to cation exchange chromatography on Hiprep CM FF 16/10 (1.6x10 cm) using Äkta Purifier HPLC system. Elution was carried out at a flow rate of 2.25 ml/min with a linear gradient of 50 mM Tris-Cl, pH 7.4 containing 0.8 M NaCl. Ion exchange fraction, CM-II with highest PLA₂ and anticoagulant activity was subjected to Rp-HPLC using Jupiter C₁₈ column (3 μ, 4.6 x 250 mm, 300 Å). The column was pre-equilibrated with milli q containing 0.1% trifluoro acetic acid (TFA) and fractionated using 80% acetonitrile (MeCN) containing 0.1%TFA on Äkta Purifier HPLC system. The homogeneity of the purified protein (5 μg of the protein treated with 0.45 μl of β-mercaptoethanol) was validated on SDS-PAGE and stained using Pierce^TM silver staining kit, Life technologies, Thermo Fischer Scientific (MA, USA) [28 (link)]. This Rp-HPLC purified protein, named as daboxin P was used for characterization in the following experiments.

For analysis of Vps75–Rtt109 only (Fig 1C), proteins were dialyzed into 20 mM Tris pH 7.5, 150 mM NaCl, and 1 mM TCEP. Mixtures were formed relative to 10 μM Vps75. Rtt109 alone was also prepared at 10 μM. 100 μl of each sample was injected over a Superdex 200 10/300 GL column using an ÄKTA purifier HPLC system (GE Healthcare) at 0.3 ml/min. This system was directly connected to a Dawn Heleos II multi-angle light scattering instrument and a refractive index detector (Wyatt Technologies). Data were analyzed with ASTRA 5.
For analysis of Vps75–Rtt109 anion exchange peaks (Fig 2E) and Vps75–Rtt109–(H3-H4) complexes (Fig 5A), samples were dialyzed into to 20 mM Tris–HCl pH 7.5, 300 mM NaCl, and 0.5 mM TCEP. Samples were concentrated to 20 μM or mixtures formed relative to 20 μM Vps75. 100 μl of each sample was injected over a Superdex 200 Increase 10/300 GL column using an ÄKTA pure HPLC system (GE Healthcare) at 0.75 ml/min. This system was directly connected to a miniDAWN TREOS and Optilab T-rEX refractive index detectors (Wyatt Technologies). Data were analyzed with ASTRA 7. Detailed protocols are published (Sarkar et al, 2020 (link)).