WangLab:Covalently Coupling Beads

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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
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