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