Hitrap nhs activated column

To prepare the BGRP column, a Hitrap NHS-activated column (Cytiva, Marlborough, MA, USA) was conjugated with supBGRP according to the manufacturer’s protocol. Before its use for BG purification, the BGRP column was washed with 10 mL of PBS that was flowed through with a peristaltic pump set at 2 mL/min. Then, 900 mL of JCP extract or 100 mL of CA sup was passed through the column at 2 mL/min. After washing the column with 10 mL of PBS (wash fluid), the BG was eluted as five fractions (900 μL/fraction) using 0.03 M NaOH. The eluates were immediately neutralized with 300 μL of 0.1 M phosphate citrate buffer (pH 3.0). After elution, the column was washed with 10 mL of PBS. Finally, the liquid in the column was replaced with PBS containing Proclin 250 (Sigma-Aldrich) and the column system was stored at 4 °C.

Preparation of the Pollen BG was followed by the procedure used in a previous paper [7 (link)]. To prepare the BGRP column, a Hitrap NHS-activated column (Cytiva, Marlborough, MA, USA) was conjugated with S-BGRP according to the manufacturer’s protocol. Before use for BG purification, the BGRP column was washed with 10 mL of PBS that was flowed through a peristaltic pump set at 2 mL/min. The pollen extract was prepared as follows. Five grams of Japanese cedar pollen (Fujifilm Wako, Osaka, Japan) were suspended in 1 L of 0.1M NaHCO₃ aqueous solution and stirred for 30 min at room temperature. Then the supernatant was collected by 2 step centrifugations with 6000 × g for 10 min and 10,000 × g for 10 min. Finally, the supernatant was filtered with a 0.20 µm aPES bottle top filter (Thermofisher Scientific, Waltham, MA, USA). Then, 900 mL of the pollen extract was passed through the column at a rate of 2 mL/min. After washing the column with 10 mL of PBS (wash fluid), the BG was eluted as five fractions (900 µL/fraction) using 0.03 M NaOH. The eluates containing Pollen BG were immediately neutralized with 300 µL of 0.1 M phosphate citrate buffer (pH 3.0), dialyzed against deionized water, and lyophilized.