Superdex 75 10 300 gl column

D. siamensis venom was dissolved in buffer A (50 mM phosphate pH 6.0) and centrifugated at 10,000 rpm for 5 min. The supernatant was loaded onto a HiTrap CM FF column (GE Healthcare, Uppsala, Sweden). The column was equilibrated with 5 volumes of buffer A and elution was carried out with an increasing linear gradient of 0–1 M NaCl in buffer A at a flow rate of 0.5 mL/min for 15 column volumes. Absorbance of the elution was monitored at 280 nm and the eluted fractions were collected using an AKTA pure Fast Protein Liquid Chromatography system (GE Healthcare, Uppsala, Sweeden). Four peaks were obtained from the elution chromatogram and each peak was tested for PLA₂ activity using the Holzer and Mackessy method [21 (link),41 (link)]. Peaks with PLA₂ activity were pooled and further fractionated using a Superdex^TM 75 10/300GL column (GE Healthcare, Uppsala, Sweden) mounted on a AKTA pure Fast Protein Liquid Chromatography system (GE Healthcare, Uppsala, Sweden). The fraction was eluted using 10 mM PBS, pH 7.4, at room temperature. The flow rate was set at 0.5 mL/min and 1 mL fraction was collected in each tube and elution was run for 50 min. The eluted proteins were detected by absorbance at 280 nm.

All size exclusion chromatography (SEC) experiments were performed in the same buffer (10 mM Hepes pH 7.4, 150 mM NaCl) using a Superdex^TM 200 increase 10/300GL column (GE-Healthcare) for YFP-NP_TAIL fusion proteins and a Superdex^TM 75 10/300GL column (GE-Healthcare) for all the other NP_TAILs. Samples were diluted to be used at 30 μM for YFP-NP_TAIL fusion proteins, 60 μM for NP_TAILs and 25 μM importin-α7 in 300 μL. They were incubated for one hour at room temperature before injection on a NGC system (Bio-Rad).

The oligomerization status of SmSOD was analyzed by gel-filtration on a Superdex^TM 75 10/300 GL column (GE Healthcare) connected to an FPLC^TM system. The column was equilibrated and eluted at 0.5 mL/min at room temperature (20–24 °C) with a 20 mM Tris•HCl buffer, pH 7.8, containing 150 mM KCl and 1% (v/v) DMSO. However, where indicated, the equilibration and elution buffer also contained 200 µM inhibitor. The protein molecular mass standards used for calibration of the size exclusion chromatography were bovine serum albumin (68 kDa), egg albumin (46 kDa), carbonic anhydrase (30 kDa) and cytochrome c (12.4 kDa).

Separation of different Aβ40 species in the presence or absence of 10 molar equivalents of DOPAL after 20 h of incubation was obtained by gel filtration using a Superdex^TM 75 10/300 GL column (GE Healthcare) connected to an ÄKTAprime (GE Healthcare) system. The column was equilibrated with phosphate buffer pH 6.9 and the elution was followed at a flow rate of 0.5 ml/min. The absorbance was measured at 280 nm.

Analytical SEC for apparent molecular weight determination of monomeric and dimeric Krm1 ECD proteins was performed on a Superdex^TM 75 10/300 GL column (GE Healthcare) connected to an ÄKTA FPLC system (GE Healthcare), which was fitted with a 1-ml sample loop. The column was pre-equilibrated with Krm1 ECD SEC buffer (50 mm Na₂HPO₄, 100 mm NaCl, pH 7.5). Samples of 3 μm Krm1 ECD were made up in Krm1 ECD SEC buffer before injecting samples (500 μl) onto the column, which was run for 2 column volumes with a flow rate of 0.6 ml/min while monitoring the A₂₈₀. A calibration was performed for the SEC column to allow the estimation of molecular weights. The calibration was done using the Gel Filtration LMW Calibration Kit (GE Healthcare). Globular protein standards were made up in 50 mm Na₂HPO₄, 100 mm NaCl, pH 7.5, at the manufacturer's recommended concentrations. The relationship between the logarithm of the molecular weight and elution volume for the globular standard was used to plot a standard curve. The resulting line of best fit was used to calculate apparent molecular weights of the Krm1 ECD species.

Size exclusion chromatography (SEC) was performed on the affinity-purified proteins using an AKTA Pure system. After centrifugation at 13,000 rpm for 5 min (to sediment any Debris), the purified protein was loaded onto a SuperdexTM 75, 10/300 GL column (GE Healthcare), preequilibrated with 25 mM HEPES (pH 8) at a flow rate of 0.3 mL/min. The time of elution of WT and Y649C was compared with a gel filtration standard containing bovine thyroglobulin (MW 670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), ribonuclease A type I-A from bovine pancreas (17 kDa) and p-aminobenzoic acid (1.35 kDa).