Immobilizing a Protein on a CM5 Chip for SPR

Materials

• 0.4M N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Make fresh in water just before use
• 0.1M N-hydroxysuccinimide (NHS). Make fresh in water just before use
• EDC/NHS Combine one part EDC with one part NHS
• 1M ethanolamine hydrochloride/NaOH pH 8.5
• 50 mM NaOH + 1 M NaCl
• 10 mM HCl + 0.1% SDS
• Water
• 10mM Glycine HCl, from ph1.8-pH2.5
• 100mM HCl
• CM5 Biacore Chip
• Protein of interest to be immobilized. Ideally start with 10-100 ug/mL protein (need ~100 uL per surface)
• Immobilization buffer (Try PBS pH 6.5 to start). Lower pH will give better immobilization, but potentially less protein function
• Experimental running buffer (Try 25 mM HEPES, 150 mM NaCl plus whatever else you need)

Considerations

• For the negative (control) surface you can immobilize either nothing (will be an ethanolamine surface) or GST or both.
• It is best to have the negative control surfaces before the ligand surfaces.
• If possible, it is good to immobilize several different concentrations of ligand, to ensure the Kd is independent of the amount of immobilized ligand.

Protocol

1. Dialyse protein extensively into PBS. You must remove all primary amines (HEPES and Tris) from the buffer.
2. Filter all solutions. and Prepare tubes of >200 uL of 50 mM NaOH + 1 M NaCl, 10 mM HCl + 0.1% SDS, Water, EDC/NHS, Ligand and Ethanolamine in plastic vials
3. Set Biacore to run at 100 uL/min
4. Inject 50 mM NaOH + 1M NaCl for 0.5 min (50 uL) twice.
5. Inject 10 mM HCl + 0.1% SDS for 0.5 min (50 uL) once.
6. Inject water for 0.5 min (50 uL) once.
7. Slow flow rate to 10 uL/min and select only the flow cell you want to immobilize on
8. Inject EDC/NHS for 7 min (70 uL)
9. Inject Ligand sample for 5 min (50 uL)
10. Inject ethanolamine for 5 min (50 uL) to block all activated groups.
11. Inject experimental running buffer for 5 min (50 uL) to allow protein to equillibrate
12. Ideally this should result in >3000 RU of immobilized ligand.
13. Repeat steps 7-12 for other ligands.