WangLab:Covalently Coupling Beads: Difference between revisions
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D. Knipmeyer (talk | contribs) (New page: == Protocols for coupling magnetic carboxyl beads to EGF or Fibronectin == *From T.M. Jovin, J. cell Science 114, p2437 (2001) ## activating beads, 0.1M sulfo-NHS 0.1M EDC RT, 1hr in 0.1M...) |
D. Knipmeyer (talk | contribs) |
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## 2x wash 0.1M MES | ## 2x wash 0.1M MES | ||
## equilibrate in coupling buffer 0.1M sodium phosphate, ph 8 | ## equilibrate in coupling buffer 0.1M sodium phosphate, ph 8 | ||
## 50 | ## 50 ug EGF (Fn?) or BSA (as control) in 30 l coupling buffer / 6 l of 5% bead slurry. Overnight rocking 4oC, coupling | ||
## 2x wash coupling buffer | ## 2x wash coupling buffer | ||
## 1M ethanolamine, RT, 2 hr, (quenching) | ## 1M ethanolamine, RT, 2 hr, (quenching) | ||
| Line 14: | Line 14: | ||
## make 1 ml 1% bead suspension in 50 mM MES at ph 6.1 | ## make 1 ml 1% bead suspension in 50 mM MES at ph 6.1 | ||
## prepare stock solutions of EDC (200mM) and NHS (500mM) | ## prepare stock solutions of EDC (200mM) and NHS (500mM) | ||
## esterification, add 10 | ## esterification, add 10 ul of the EDC and NHS stock solutions to the beads (2mM EDC and 4mM NHS). Incubate 15min while rocking | ||
## prepare 1ml sodium bicarbonate buffer at ph 8.3, add EGF to a final conc | ## prepare 1ml sodium bicarbonate buffer at ph 8.3, add EGF to a final conc 1ug/ml | ||
## Quench EDC, add 1, | ## Quench EDC, add 1,4ul BME to the beads after the incubation | ||
## wash the beads, quick | ## wash the beads, quick | ||
## couple the EGF, put the beads in the EGF solution at ph 8.3 | ## couple the EGF, put the beads in the EGF solution at ph 8.3 | ||
## incubate at RT for 30 min | ## incubate at RT for 30 min | ||
## Quench reaction, add10 | ## Quench reaction, add10 ul 1M hydroxylamine | ||
## thorough wash with PBS | ## thorough wash with PBS | ||
## store in 50%glycerol at –20oC | ## store in 50%glycerol at –20oC | ||
Latest revision as of 11:11, 20 June 2007
Protocols for coupling magnetic carboxyl beads to EGF or Fibronectin
- From T.M. Jovin, J. cell Science 114, p2437 (2001)
- activating beads, 0.1M sulfo-NHS 0.1M EDC RT, 1hr in 0.1M MES (ph5)
- 2x wash 0.1M MES
- equilibrate in coupling buffer 0.1M sodium phosphate, ph 8
- 50 ug EGF (Fn?) or BSA (as control) in 30 l coupling buffer / 6 l of 5% bead slurry. Overnight rocking 4oC, coupling
- 2x wash coupling buffer
- 1M ethanolamine, RT, 2 hr, (quenching)
- 2x thorough wash with PBS
- store in PBS+0.1% sodium azide
- from Philippe Bastiaens, Science, EGFR activation.
- make 1 ml 1% bead suspension in 50 mM MES at ph 6.1
- prepare stock solutions of EDC (200mM) and NHS (500mM)
- esterification, add 10 ul of the EDC and NHS stock solutions to the beads (2mM EDC and 4mM NHS). Incubate 15min while rocking
- prepare 1ml sodium bicarbonate buffer at ph 8.3, add EGF to a final conc 1ug/ml
- Quench EDC, add 1,4ul BME to the beads after the incubation
- wash the beads, quick
- couple the EGF, put the beads in the EGF solution at ph 8.3
- incubate at RT for 30 min
- Quench reaction, add10 ul 1M hydroxylamine
- thorough wash with PBS
- 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
