Cosmospin filter h

The toxic strain of the freshwater cyanobacterium A. circinalis used in this study is a nonaxenic strain TA04. The field sample of A. circinalis was collected at the Tullaroop reservoir, Victoria, Australia, and the TA04 strain was one of single-trichome isolates prepared by Negri et al. [33 (link)]. A. circinalis (TA04) was provided by Dr. Susan Blackburn, CSIRO, Australia, and cultured in CB’ medium [see, ref.12, SI] (30 mL). The cells were harvested by filtration using a glass fiber filter (GA100, 1.0 μm, Advantec, Tokyo, Japan), suspended with 2.0 mL of 0.5 M AcOH and sonicated three times for 30 s on ice. Then, the resulting solutions were centrifuged at 20,000× g for 5 min at 4 °C. The supernatants were filtered through a Cosmospin filter H (0.45 μm, Nacalai Tesque, Kyoto, Japan). A part (200 μL) of the filtrate was adjusted to pH 7–8 using 2 M NH₃ aqueous, and loaded on an activated charcoal column (100 μL vol.) (for chromatography, 63–300 µm >40%, Wako Pure Chemical Industries, Ltd.). After the column was washed with water (0.2 mL), PST were eluted with AcOH/EtOH/H₂O (5:50:45, v/v/v, 1 mL). The solvent was removed using N₂ gas, and the resulting residue was resuspended with 100 μL of 0.05 M AcOH.

Purified antibodies were filtered using Cosmo spin filter H (Nacalai Tesque, Inc.) to remove precipitates or debris before SEC analysis. Then, the antibodies were passed through an Agilent Bio SEC-5 500 Å (7.8×300 mm) column (Agilent Technologies) coupled to an Agilent 1260 Infinity Bio-inert HPLC system (Agilent Technologies). Analyses were performed using 1 μg or more of each antibody sample, with a flow rate of 1 ml/min. PBS (pH 7.4) was used as an eluent. Data were analyzed using OpenLAB CDS ChemStation Edition (Agilent Technologies).