Long arm biotin labeling kit

The binding affinity between FMBP and GRP78 was determined by biolayer interferometry technology using the OctetRED system (ForteBio). Purified FMBP was biotinylated by Long-arm Biotin Labeling Kit (Elabscience Biotechnology Co., Ltd.) following the manufacturer's protocol. Bio-FMBP (2 mg/mL) was then incubated with super-streptavidin biosensors in binding buffer (20 mmol/L HEPES pH 7.4, 150 mmol/L NaCl) and washed three times in binding buffer. Recombinant GRP78 was serially diluted by binding buffer, and the association/dissociation of FMBP:GRP78 was monitored by OctetRED for 10 min at 25 °C. Data were analyzed using Octet Data Analysis Software 7.0 (ForteBio).

Purification of FMBP was performed according to our previous described method¹⁹ (link). Foxtail millet bran was soaked in deionized 0.02 mol/L Tris-HCl buffer for 24 h, and then the supernatant was precipitated with 80% saturated ammonium sulfate at 4 °C for 30 min, followed by purification with SP cation-exchange column. The eluted fraction of 100 mmol/L NaCl was collected and incubated for 20 min at 80 °C and showed a single band on SDS-PAGE, named FMBP.
FMBP was treated with Long-arm Biotin Labeling Kit (Elabscience Biotechnology Co., Ltd.) according to the manufacturer's instructions, and then the Bio-FMBP was obtained. Briefly, 1 mg of FMBP was dissolved in 0.5 mL label buffer and added into ultrafiltration tube. After centrifuged 12,000 × g for 10 min, appropriate NH₂-Reactive Biotin and labeling buffer were added to the filtration tube with gently blowing blending. The mixture was incubated in darkness at 37 °C for 30 min, followed by centrifugation at 12,000 × g for 10 min, and then washed twice with labeling buffer. The ultrafiltration tube was inverted in a new EP tube and centrifugated at 6000 × g for 10 min. The biotin-labeled FMBP solution was collected and kept at 4 °C.