Loading FGF beads

L. Maves 100703

I. Beads: Polystyrene microspheres from Polysciences. I use 45 micron; they have many different sizes.

II. FGF protein can be ordered from R&D Systems. I use recombinant mouse FGF-8b. Reconstitute according to R&D instructions: Dissolve protein in 100 ul of filter-sterilized (f/s) PBS with 0.1% BSA to make a 250 ug/ml stock. Aliquot to 10 ul and store at ≠80C.

III. Load beads the day of your experiment (I havenπt experimented with using old beads). Add a drop of bead solution to a 1.5 ml tube. Pulse spin down the beads. Pipette off water. Add 100 ul f/s PBS (no BSA) to rinse. Spin down and repeat again for 2 PBS washes. Spin down and pipette off PBS. Add 20 ul of 0.5 mg/ml Heparin in f/s PBS to pre-coat beads. Let beads incubate in heparin for 20 min at RT-keep tubes upright on a flat table shaker. Spin down beads and pipette off heparin. Add about 10 ul of FGF protein stock solution-incubate again for 2 hours (incubate in PBS with 0.1% BSA for control beads). Spin down beads, pipette off protein solution, add 50 ul of f/s PBS to rinse. Keep beads chilled until ready to use.