WangLab:Coupling Carboxyl Beads to EGF or Fibronectin

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