WangLab:Coupling Carboxyl Beads to EGF or Fibronectin

From T.M. Jovin, J. cell Science 114, p2437 (2001)

 * 1) 2x wash 0.1M MES at 4oC
 * 2) activating beads (RT, 1hr)
 * 3) *0.1M sulfo-NHS
 * 4) *0.1M EDC
 * 5) *in 0.1M MES (ph5)
 * 6) 2x wash 0.1M MES at 4oC
 * 7) equilibrate in coupling buffer 0.1M sodium phosphate, ph 8
 * 8) 10-50 ug EGF (Fn?) or BSA (as control) in 300 ul coupling buffer / 30 ul of 10% bead slurry. Overnight rocking 4oC, coupling
 * 9) 2x wash coupling buffer (centrifuge 1,6000 g x 3 min at 4 oC)
 * 10) 1M ethanolamine, RT, 2 hr, (quenching)
 * 11) 2x thorough wash with PBS (centrifuge 1,6000 g x 3 min at 4 oC)
 * 12) store in PBS

Materials:

 * sera-mag beads 0.768 um, 5% slurry, seradyn.com, cat#294766050250
 * 500mM MES, ph 5.0, store 4oC, Fw 195.2=9.76g/100ml
 * 500 mM NHS, make fresh, Fw115=575mg/10ml, powder in 4oC
 * 500 mM EDC, make fresh, Fw192=960mg/10ml, powder in –20oC
 * 500 mM Na2HPO4 ph 8.0, Fw 142=7.1g/100ml, ph w/HCl
 * 1M ethanolamine Fw61.08=611mg/10ml

From Philippe Bastiaens, Science, EGFR activation.

 * 1) make 1 ml 1% bead suspension in 50 mM MES at ph 6.1
 * 2) prepare stock solutions of EDC (200mM) and NHS (500mM)
 * 3) esterification, add 10 ul of the EDC and NHS stock solutions to the beads(2mM EDC and 4mM NHS). Incubate 15min while rocking
 * 4) prepare 1ml sodium bicarbonate buffer at ph 8.3, add EGF to a final concentration 1ug/ml
 * 5) Quench EDC, add 1,4ul BME to the beads after the incubation
 * 6) wash the beads, quick
 * 7) couple the EGF, put the beads in the EGF solution at ph 8.3
 * 8) incubate at RT for 30 min
 * 9) Quench reaction, add10 ul 1M hydroxylamine
 * 10) thorough wash with PBS
 * 11) store in 50%glycerol at –20oC