WangLab:Covalently Coupling Beads

Protocols for coupling magnetic carboxyl beads to EGF or Fibronectin

 * From T.M. Jovin, J. cell Science 114, p2437 (2001)
 * 1) 	activating beads, 0.1M sulfo-NHS 0.1M EDC RT, 1hr in 0.1M MES (ph5)
 * 2) 	2x wash 0.1M MES
 * 3) 	equilibrate in coupling buffer 0.1M sodium phosphate, ph 8
 * 4) 	50 ug EGF (Fn?) or BSA (as control) in 30 l coupling buffer / 6 l of 5% bead slurry. Overnight rocking 4oC, coupling
 * 5) 	2x wash coupling buffer
 * 6) 	1M ethanolamine, RT, 2 hr, (quenching)
 * 7) 	2x thorough wash with PBS
 * 8) 	store in PBS+0.1% sodium azide


 * 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 conc 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

Materials

 * sera-mag beads 0.768 m, 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