Human fgf21

FGFR1–KLB–bFKB1 interaction was studied by SPR measurements on a ProteOn XPR36 instrument (Bio-Rad) at 25 °C. FGFR1–ECD (extra-cellular domain) protein (20 μg per ml) at pH 4.5 was immobilized at surface density (1000 RU) on an activated ProteOn GLC sensor chip using standard amine coupling procedures as described by the manufacturer. bFKB1 or 1:1 mixtures of bFKB1 and KLB–ECD were injected in PBS containing 0.005% v/v Tween-20, 0.3 M NaCl (pH 7.4) at a flow rate of 80 μl per min and sensorgrams for association and disassociation phases were recorded. Analytes were injected for 5 min and allowed to disassociate for 10 min. Data was referenced with interspots, processed, and disassociation constants measured with the ProteOn Manager software (version 3.0, Bio-Rad). bFKB1–KLB–FGF interaction was studied by bio-layer interferometry (BLI) measurements on an Octet Red instrument (ForteBio) at 25 °C. bFKB1 (20 μg per ml) at pH 4.5 was immobilized on activated amine reactive biosensor tips as described by the manufacturer. KLB–ECD (20 μg per ml) in PBS containing 0.005% v/v Tween-20, 0.3 M NaCl (pH 7.4) was captured onto the same biosensor tips and measured with human FGF21 (R&D Systems) in the same buffer. Qualitative data was processed with the data acquisition software (ForteBio).