Activation & Capture (CSPR)


This protocol is used to activate a pre-conditioned CM sensor chip for amine coupling. The sensor chip can be used as is for amine coupling of ligands or strepavidin or neutravidin can be coupled to make a SA (NA) chip for the capture of biotinylated compounds.

You will need:

  • a fresh CM chip which has been pre-conditioned
  • N-hydroxysuccinimide (NHS) which has been diluted and aliquoted. Individual vials are found in the freezer
  • 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which has been diluted and aliquoted. Individual vials are found in the freezer.
  • An empty vial
  • Ligand or strepavidin/neutravidin. DO NOT PREPARE IN ACETATE BUFFER UNTIL IMMEDIATELY BEFORE USE!
  • For strepavidin, prepare immediately before use, 2 mg/ml (1 mg/500 µl acetate buffer) solution of strepavidin in acetate buffer, pH 4.5. Centrifuge in table top ultracentrifuge at 100,000 rpm, 4°C, for 10 min. Avoiding any pellet, transfer immediately to a 7 mm vial and cap.

Procedure:

  • Dock the new chip and prime 3X with running buffer.
  • Because of the time sensitive nature of activation and capture, flow cells are done individually if different ligands are to be captured on individual flow cells or if different RU values of the same ligand are to be captured on different flow cells. Generally Flow Cell 1 (FC1) is activated and blocked immediately and used as a control surface (see figure above). Run:Start Sensorgram and choose the appropriate flow cell. The rate is set to 5 µl/min. The NHC and EDS are aliquoted and frozen. Immediately prior to use, remove one tube of each and defrost and gently mix. Place in R2E1 and R2E2 and place an empty tube in R2E3. The NHC and EDS are diluted 50:50 by the instrument and the mix is placed in the empty tube. Go to Command:Dilute and enter the information from the picture below and then click “OK”.

  • In the Command Queue enter Inject: Quickinject and enter a 35 µl volume which allows 7 minutes of contact time.

  • If you are going to use this flow cell surface as a reference, pipet 1 ml of 1 M ethanoloamine into a 16 mm glass vial, cap, and place in R2F3. Select Command:Inject:Quickinject and set the volume to 35 µl. Click on More and select Extraclean; begin injection.
  • For surfaces on which ligand is to be coupled, activate as described. Close the command queue. Select Command:Inject:Manual Inject. Load the loop. Manually inject small volumes such as 2 µl to see what response you get. Use the flags to mark on the sensorgram the baseline and to note injections. Once you have achieved the desired RU, close the manual inject and empty the loop. Block the surface following the procedure above by injecting 1 M ethanolamine over the surface with the extra cleanup step selected. Set the baseline after activation and determine the total RU of the surface after blocking. The surface below has approximately 2000 RU of protein bound to the surface after blocking.
  • For preparing a strepavidin chip, open all 4 flow cells and activate as above. Upon activiation of the 4 surfaces, under Commands:Inject:Manual Inject and fill the loop with 100 µl of clarified strepavidin. Inject until the desired level is reached (5,000 – 10,000 RU). Block with ethanolamine as described above. Unbound strepavidin can be removed with 5 – 7 injections of 1 M NaCl/50 mM NaOH for 1 minute each at 20 µl/min.