Protein a beads

To study the interaction between Fc-tagged FGFR isoforms, FGF isoforms and sKL, we conducted precipitations studies. Protein A beads (6501, Biovision) and protein G beads (6511, Biovision) were used at 25 μL of a 50% stock slurry per sample, in a total of bead volume of 50 μL. Beads were washed 5x in activity buffer (50 mM Tris pH 7.4, 200 mM NaCl, 0.01% Tween 20). Bead spin downs were all performed for 2 minutes at 5,000 g. Beads were resuspended in 1 mL of activity buffer in a 1.5 mL snap cap tube. 1 μg of Fc-tagged FGFR1–4 were incubated with the beads for 1 hour on a bead rotator. Control samples were incubated with 2 μg of anti-FGF antibody instead of Fc-FGFR. Beads were then pelleted at 5,000 g for 2 minutes, and washed 5x with activity buffer. Beads were resuspended and incubated with different combinations of 500 ng of FGF, 1 μg of sKL, 1.6 USP heparin, or PBS. Beads were incubated for 1 hour on a tube rotator and beads were pelleted and washed as before. 100 μL of 1x Laemmli sample buffer (1610737, Biorad) with 1.42 M 2-mercaptoethanol added and boiled for 5 minutes. Samples were analyzed by SDS-Page and Western blotting.