Gel filtration lmw calibration kit

To determine whether the disulfide bonds exist in FBN30, the non-reducing (without β-ME) and reducing conditions of 12% SDS-PAGE gel were performed and the expression of FBN30 in the cellular pellet and medium were detected by using anti-FBN30 antibodies. To check the molecular weight of the native FBN30, which may form oligomers, proteins were further separated using size exclusion chromatography. Samples were applied onto a Superdex 200 10/300 GL size exclusion column (GE, Fairfield, CT) on an AKTA Pure FPLC system (GE). The column was pre-calibrated to generate a standard curve with a set of proteins differing in size (Gel Filtration LMW Calibration Kit, GE). The chromatography elution buffer used was phosphate buffered saline (PBS, 3 mM sodium phosphate dibasic, 1mM potassium phosphate monobasic, 155 mM sodium chloride, pH 7.4, Life Technology) and the sample volumes were set at 500 µL. The elution was carried out using isocratic elution at a flow rate of 0.4 mL/min and the fractions were collected 100 µL per fraction. FBN30 protein in each elution fraction was measured by using polyclonal anti-FBN30 antibodies in ELISA.

Gel filtration was performed on a Superdex S75 16/600 column (Pharmacia Biotech) with buffer containing 50 mM Tris pH 8.0 and 100 mM NaCl at a flow rate of 1 ml/min. The protein complex solution was injected into the column at a final concentration of 1 mg/ml in a total volume of 1 ml. Fractions were analyzed by SDS-PAGE and Coomassie blue staining. For protein molecular weight determination, the column was calibrated with a gel filtration LMW calibration kit (GE Healthcare) using the running buffer described above.

The supernatant of the transfected cell cultures were collected a fortnight later and spun down at 4,000 rpm, 4°C, for 1 h before 0.2 µm filtration and purified using the ÄKTA pure system (GE Healthcare) using Protein G affinity column for the IgGs as previously described (15 (link)). The 5 ml HiTrap TALON crude affinity column (GE Healthcare) was used for the recombinant FcγIIA using the same settings. The affinity purified proteins were subsequently gel filtrated using the Superdex 200 pg 16/60 column (GE Healthcare) precalibrated with Gel filtration HMW calibration kit (GE Healthcare) and the Gel filtration LMW calibration kit (GE Healthcare) to extract the monomeric fractions as previously described (14 (link), 15 (link)). Pure IgG1 variants and FcγIIA fractions were collected and concentrated using 100 kDa Amicon Pro System (Merck) and 10 kDa Amicon Pro System (Merck) concentrators, respectively. The final concentrations of the proteins were determined by spectrophotometric analyses using nanodrop (Thermo Fisher Scientific) with consideration of calculated protein extinction coefficients of the various IgG1 variants and FcγIIA.

The oligomeric state and homogeneity of xylanase 1 was determined by size-exclusion chromatography on Superdex 75 (10/30) column (GE Healthcare, Canada), equilibrated with McDougall’s buffer, pH 6.0. Molar mass of the protein peak was calculated using a logarithmic interpolation of elution volumes (Ve) using a gel filtration LMW calibration kit (GE Healthcare, Pittsburgh, USA) containing (1) blue dextran 2000 (V₀), (2) thyroglobulin (670 kDa), (3) g-globulin (158 kDa), (4) ovalbumin (44 kDa), (5) myoglobulin (17 kDa), and (6) vitamin B12 (1.3 kDa).

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.

Analytical size exclusion chromatography (SEC) was performed at a flow-rate of 0.5 ml/min using a Superdex 75 10/300 column (GE Healthcare Life Sciences) pre-equilibrated in 20 mM TrisCl, 150 mM NaCl, 1 mM EDTA pH = 7.9. The column was calibrated with a separate run of appropriate globular marker proteins (Gel Filtration LMW Calibration Kit, GE Healthcare Life Sciences).

Analytical size exclusion chromatography was carried out as described previously 43, 44 on a Micro‐Äkta purifier system equipped with Superose 6 Increase 10/300 GL column (GE Healthcare, Freiburg, Germany). The Superose column was coupled to a triple‐angle light scattering (MALS) detector (MiniDAWN^™ TREOS^® system; Wyatt Technology Corp., CA, USA) and a refractometer (Optilab T‐rEX, Wyatt). The column was calibrated using standard proteins: thyroglobulin (670 kDa), ferritin (440 kDa), globulin (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), RNase (13.7 kDa) (Bio‐Rad gel filtration standard, GE Healthcare LMW gel filtration calibration kit). Bovine serum albumin (BSA) was used to calibrate and validate the MALS analysis. The running buffer consisted of 10‐mm Tris pH 7.8, 300‐mm NaCl, 2‐mm MgCl₂, 0.02% NaN₃ and 2% glycerol. The samples were centrifuged for 5 min at 18 407 g, and 100 μL of the supernatant were injected for analysis with a flow rate 0.5 mL·min⁻¹. The resulting data were analysed with ASTRA program (Wyatt Technology, Dernbach, Germany). The elution volume was plotted against the UV signal and molecular weight profiles. The apparent molecular weights were derived from MALS data. The chromatographic elution profiles were collected (0.5‐mL fractions) and analysed by Glycine‐SDS/PAGE.