Recombinant human fap

Recombinant human FGF-21 (Peprotech) or recombinant mouse FGF-21 (ProSpec Protein Specialists) was reconstituted in FAP assay buffer (50 mM Tris, 140 mM NaCl, pH 7.5). Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099. For SDS-PAGE analysis, samples were immediately added to 2x gel loading buffer (0.6 ml 1M Tris pH 6.8, 2.5 ml 50% glycerol, 2 ml 10% SDS, 1 ml 1% bromophenol blue, 3.4 ml H20 and 0.25 ml β-mercaptoethanol/ 5.5 ml aliquot). 3 μg of protein was then loaded onto a reducing 20% SDS-PAGE gel. Gels were stained with Gelcode Blue Stain Reagent (Thermo Scientific). Alternatively, for LC/MS, aliquots of the reaction were taken and quenched with 10% v/v .01 M HCL and run on 1100 series LC/MSD (Agilent and HP). LC solvents were H₂O+.01% TFA (solvent A) and acetonitrile+.08% TFA (solvent B). LC was set to 2% solvent B 0–2 minutes followed by 40–88% solvent B gradient from 2–30 minutes (Column: Zorbax C-18, 2.2 x 50 mm, 3.5 μM). Percent cleavage of FGF-21 was quantified by extracted ion chromatogram integration of peaks corresponding to the +10, +11 and +12 ions of both cleaved and intact FGF-21. The half-life was calculated using one phase decay function on GraphPad Prism software.