Frac30 fraction collector

The total proteins 7,8,10 of 5 g transgenic hairy root were extracted using phosphate buffer (100 mM, pH 7). First, the hairy root clones were ground under liquid nitrogen, and the powder was suspended in 1:1 phosphate buffer w/v. The recombinant protein was purified using low pressure chromatography system from GE Healthcare Life sciences (ÄKTA start, UNICORN start, and Frac30 fraction collector). Initially a HisTrap FF crude, 1 x 5 ml (GE Life-sciences) column was equilibrated by loading 600 μL of lysis buffer (25 mM NaH2PO4, 150 mM NaCl, 10 mM imidazole, pH 8.0). Next, up to 800 μL of concentrated root extract was loaded onto the column through the system pump. Then, the column was washed twice with 600 μL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). Finally, the protein was eluted twice using 300 μL of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0) and collected1 ml fractions using Frac30 fraction collector at 1 ml/min flow rate and 80% step gradient method at 280nm. Then imidazole removed from pooled fractions using a 5 ml HiTrap™ Desalting column with the predefined Desalting template available in UNICORN start. Further purity of the protein analyzed the using SDS-PAGE, and ELISA immunodetection (BioEra Life Sciences) to confirm activity and specificity of the protein.

Spirulina tablets cultured from lava seawater were donated by KIOST (Gyeonggi-do, Korea). Piperazine, sodium chloride, and C-PC (spirulina sp.) were purchased from Sigma Aldrich (USA).
Tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), disodium salt dihydrate, and sucrose were purchased from USB Corporation (USA). Ammonium sulfate and hydrochloric acid were purchased from DaeJung Chemical Co. (South Korea) and Samchun Chemical Co.
(South Korea), respectively. The following methodologies and relevant kits were used in this study: centrifugation (Supra 25K, Hanil, South Korea); anion exchange chromatography (HiTrap Q HP, GE Healthcare, USA); dialysis (MEMBRA-CEL MC18 X 100 CLR; Serva, Germany); AKTA start puri cation system with Frac30 fraction collector (GE Healthcare, USA); spectroscopy with a microplate reader, equipped with a 50 W xenon ash lamp (Varioskan LUX, Thermo Scienti c, USA); RNeasy Mini kit plus (Takara Bio, Japan); QuantiFasT Reverse Transcription kit (Qiagen, Germany); Rotor-Gene Q PCR (Qiagen, Germany); ultra-centrifugation (Vivaspin 500, MWCO 10000, Sartorius Germany)