Hoatlin:Crosslinking antibodies to beads

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Revision as of 17:42, 2 January 2008 by Alexis (talk | contribs) (New page: # 400μL Protein A Sepharose Fast Flow Beads (GE Healthcare #17-1279-01) # Rotate @ room temp (RT) 30 min-3 hrs (3 hrs @ 4°C) # Wash 4X in PBS (1ml for each wach) # Wash 2x in 100mM Na B...)
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  1. 400μL Protein A Sepharose Fast Flow Beads (GE Healthcare #17-1279-01)
  2. Rotate @ room temp (RT) 30 min-3 hrs (3 hrs @ 4°C)
  3. Wash 4X in PBS (1ml for each wach)
  4. Wash 2x in 100mM Na Borate, pH 9.0
  5. Resuspend beads in 1 ml 100mM Na Borate, pH 9.0 + 5.2mg DMP (Dimethyl pielimidate, Pierce #21667)
  6. Rotate 45 min (don't exceed 1 hr) at RT
  7. Spin 1 min at RT and recover beads
  8. Wash beads 1x w/ 1M ethanolamine, pH 8.0 (Sigma #E9508)
  9. Add 1 ml of 1M ethanolamine, pH 8.0
  10. Rotate 2 hrs-overnight at 4°C
  11. Wash 4x quickly in 100 mM glycin pH 3.0
  12. Neutralize by washing 2x w/ PBS
  • Store at 4°C
  • Beads can be used several times
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