Protocol: Pre-concentration of Ligand

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 above 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.