Pollen BG Purification from Cedar Pollen

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.