Preconcentration is an important strategy for optimizing your SPR experiments. As we explained in detail in our previous post, preconcentration is used with COOH sensor chips, when ligands are covalently coupled to the chip surface using amine coupling. Preconcentration increases the local concentration of ligand at the surface by modifying the pH of the immobilization buffer to be below the the pI of the ligand, which causes an electrostatic attraction of the ligand to the surface. This increases the efficiency of ligand binding, reducing the amount of ligand needed, saving precious protein sample.
Although our amine coupling kit contains an immobilization buffer that is optimal for many ligands, it can be beneficial to test a number of different pHs to find the one that is optimal for your specific ligand. To make the buffer scouting process as efficient as possible, we recommend following the steps below to find the optimal immobilization buffer for your ligand. Using the process outlined, you don’t need to use a new sensor chip for each buffer pH tested. An unactivated COOH sensor chip is used to test the preconcentration buffers by using a regeneration solution in between each buffer, so only 1 chip is needed.
Optimal immobilization buffer conditions produced the largest signal increase due to the high and fast preconcentration of ligand on the sensor surface
1. Purchase the Immobilization Buffer Optimization Kit from our online store, which contains acetate buffer at the following pH values: 4.0, 4.5, 5.0, 5.5
2. Setup your SPR instrument with a COOH sensor chip and your running buffer of choice
3. Dissolve your ligand at a concentration of between 5-25μg/ml in each of the pHs of the acetate buffer
4. Prepare a regeneration solution (for example, 10mM HCl) which will be used to remove the ligand from the COOH surface
5. Inject the ligand sample in the pH 5.0 buffer at 20μL/min.
6. After the ligand sample has passed through the flow cell, regenerate the surface by injecting the regeneration buffer at 100μL/min.
7. Repeat step 5 and 6 with all of the different acetate buffers
8. The optimal immobilization buffer is the one at the highest pH that produces a high and fast preconcentration, as indicated by a large signal increase due to the preconcentration of ligand on the sensor surface.
The reason you want to use the highest pH that gives good preconcentration is because the EDC/NHS coupling reaction is most efficient at higher pHs.
Reference: SPR pages, “Preconcentration.” http://www.sprpages.nl/immobilization/ligand-pre-concentration, 1998.