Aktaprime plus chromatography system

A 700mg aliquot of endosperm was extracted in 1ml of gel filtration buffer containing 10mM HEPES-KOH, pH 7.5, 100mM NaCl, 10 μl ml^–1 plant protease inhibitor cocktail (Sigma) and centrifuged at 20 000 g for 10min. The supernatant was filtered through 0.45 μm cellulose acetate to remove large particles and injected into a 500 μl sample loop, prior to fractionation by gel permeation chromatography (GPC) using Superdex 200 resin packed in a 10/300 column connected to an AKTAprime plus chromatography system (GE Healthcare) at 4 °C. The column was equilibrated with 10mM HEPES-KOH, pH 7.5, 100mM NaCl, and fractions eluted at 1ml min^–1. Fractions of 2ml were collected and concentrated 25-fold using an Amicon Ultra 50K centrifugal filter unit (Merck Millipore) following the manufacturer’s instructions. Concentrated samples were mixed with one-third volume of native-PAGE sample buffer (0.625M TRIS-HCl, pH 7.0, 50% glycerol, 0.2% bromophenol blue). A 7.5 μl aliquot was applied per lane to the native (non-denaturing) PAGE (see next section). The residual samples were further supplemented with one-third volume of SDS–PAGE sample buffer (0.1M TRIS-HCl, pH 6.8, 10% SDS, 12% β-mercaptoethanol, 20% glycerol, 0.2% bromophenol blue), boiled, and 5 μl per lane subjected to 7.5% acrylamide SDS–PAGE (height 6cm, width 8.5cm, and thickness 1mm) at 25 mA, and western blotting.

A 50 mg sample of the B.moojeni venom was used to purify the metalloproteinase BmooMPα-I. The sample was resuspended in 1 mL of 50 mM ammonium bicarbonate buffer pH 8.0. The mixture was centrifuged at 13,000× g for 5 min, and then the supernatant was applied to a CM-SEPHAROSE FF column. The run was performed in the Akta Prime Plus Chromatography system (GE, Uppsala, Sweden) using a gradient (0–100%) of ammonium bicarbonate 500 mM pH 8.0 at a flow of 1 mL/min. The elution was monitored at 280 nm, and the fractions containing the BmooMPα-I were manually collected, lyophilized, and stored at −70 °C.
The isolated fraction, obtained by ion-exchange chromatography, was reconstituted in 0.1% Trichloroacetic acid. The mixture was applied to a C-18 column (Supelco). The reverse-phase chromatography was performed under a concentration gradient (0–70%) of acetonitrile 99.9% at a 1 mL/min flow in the Akta Prime Plus Chromatography system. Elution was monitored at 280 nm, and fractions were manually collected

Purple corn pericarp was extracted using a dry milling procedure [35 ]. An ASE 300 Accelerated Solvent Extraction System (Thermo Scientific) was used to extract anthocyanin from purple corn pericarp, using only water as solvent. Corn pericarp (about 10 g) was added to a stainless steel cell with cellulose filter. The cell was preheated for 5 min and heated up to 50°C for extraction; static solvent extraction time was 5 min. The pressure in the cell during extraction was 1500 psi. The amount of water to flush through the cell following the static heating step was 100% of the cell volume. Extraction cycle to perform the static heating and flushing steps was five times. Cells were purged with nitrogen for 60 s at the end of the extraction. After freeze-drying, the extracts were passed through a Sephadex® LH-20 resin column (25 x 1.7 cm) using only water as eluent in an AKTAprime plus chromatography system (GE Healthcare, Milwaukee, WI). The volume of the water extracts was reduced by 60% in a rotational evaporator, freeze-dried, and kept at -20°C until further use. The resulting powder was called purple corn anthocyanin-rich water extract (PCW).