Pre-conditioning

In order to hydrate and clean the surface of a new sensor chip, a pre-conditioning step is performed before it is used.

You will need 250 µL of each of the following solutions pipeted into a 7 mm plastic vial and capped properly.

  • 100 mM HCl
  • 50 mM NaOH (BR100358)
  • 0.5% SDS (from BIAMaintenace kit stored at room temp)

Procedure:

  • Place the vial containing the 100 mM HCl in R2A1, the vial containing the 50 mM NaOH in R2A2, and the vial containing the 0.5% SDS in R2A or in A1, A2, and A3 of the Sample and Reagent Rack of the T100.
  • From the menu, select Run:Run sensorgram. Select all four flow cells (1,2,3,4). On the T100 select Manual Run. Set the flow rate to 100 µL/min
  • Once the sensorgram starts to run, a command queue will appear on the 3000. On the T100, continue through the steps required.
  • From the menu, select Command:Inject and select Quickinject. Select the position of the 100 mM HCl (R2A1) and select the volume to be injected (10 µL) for a 6 second contact time. In the same box, click on More and check the “Extraclean” box. Click on start and the command should appear in the command queue. Repeat this a second time so that 100 mM HCl is injected 2X. On the T100, use the regeneration injection to inject 10 µL from A1, repeat, inject from A2 twice and then from A3 twice.
  • From the menu, select Command:Inject and select Quickinject. Select the position of the 50 mM NaOH (R2A2) and select the volume to be injected (10 µL) for a 6 second contact time. In the same box, click on More and check the “Extraclean” box. Click on start and the command should appear in the command queue. Repeat this a second time so that 50 mM NaOH is injected 2X.
  • From the menu, select Command:Inject and select Quickinject. Select the position of the 0.5% SDS (R2A3) and select the volume to be injected (10 µL) for a 6 second contact time. In the same box, click on More and check the “Extraclean” box. Click on start and the command should appear in the command queue. Repeat this a second time so that 0.5% SDS is injected 2X.
  • After all injections have been completed, select Run:Stop sensorgram from the top menu. Pre-conditioning has been completed and your sensorgram should be similar to the one shown above.


    pH scouting

    This protocol is used initially to determine the best pH at which to couple ligand to the surface using an unmodified CM chip (not applicable for NTA and SA sensor chips). Under low ionic strength and some pH lower than the isoelectric point of the protein, the protein will become electrostatically attracted to the surface matrix. The highest pH at which protein is attracted to the surface is chosen. Coupling is generally accomplished at lower pH’s in acetate buffer. Subsequent experiments using the same protein should not require additional pH scouting.

    You will need:

    • a fresh CM chip
    • relatively concentrated ligand so as to minimally dilute the buffer
    • 100 µl of 10 mM acetate buffer at pHs 5.5, 5.0, 4.5, and 4.0

    Procedure:

    • Dock the new chip and prime 2X with running buffer.
    • From the menu, select Run:Run sensorgram. Set the flow rate to 5 µl/min
    • Once the sensorgram starts to run, a command queue will appear.
    • Pipet 100 µl of each acetate buffer into a 7 mm plastic tube and add 5 µg of ligand immediately before use. Cap and place in R2A1 – R2A4.
    • From the menu, select Command:Inject and select Quickinject. Select the position of the first tube (R2A1) and select the volume to be injected (5 µl) for a 60 second contact time. Click on start and the command should appear in the command queue.
    • Repeat the steps above for positions R2A2, R2A3, and R2A4.
    • A sensorgram such as the one below will be shown on the screen. Choose the highest pH at which there is electrostatic attraction of the ligand with the surface, in this case pH 5.0. At pH 5.5, there was no apparent attraction and while there was attraction at both pH 4.5 and pH 4.0, these pH’s are lower than pH 5.0 and are not chosen.