Surface plasmon resonance (SPR) is the most commonly used technique to determine accurate binding kinetics. One of SPR’s advantages over other techniques like ITC is that it only requires a small amount of sample. But as with any technique, there are tricks to getting the most our of your experiment and the instrument. Preconcentration is a great method to increase the concentration of your ligand at the surface of the sensor. This allows researchers to use even less ligand when conducting SPR experiments. Since expressing and purifying proteins is time-consuming and purchasing proteins can get expensive, saving as much ligand as you can is always ideal.
A lot of users were asking us to explain preconcentration, so we put together a blog post to help out!
Preconcentration is used to increase the concentration of the ligand at the surface of the sensor
Utilized during covalent coupling to increase ligand density
Increases local concentration of ligand via electrostatic forces
What are the Benefits?
Increased efficiency of protein immobilization on sensor chip surface
Decrease in level of protein required for injection
Preconcentration is independent of the ligand concentration
What is preconcentration?
Preconcentration is a covalent immobilization method that can be used to obtain a high ligand concentration at the sensor chip surface prior to the injection of analyte. This technique can allow a localized ligand concentration greater than 100 mg/mL, even with a very low initial concentration. Preconcentration has significant benefits for carboxyl sensor chips, but limited effectiveness for chips based on ligand binding to pre-immobilized tags. Preconcentration uses electrostatic forces at the surface of the sensor chip to increase the local concentration of ligand at the reaction site on the sensor surface. This is typically used when large ligands, such as proteins, need to be immobilized to carboxyl sensor chips. A very high local concentration of ligand can be achieved at the surface, even with a very low initial ligand concentration (typically 10-100 µg/mL) Some ligands are unsuitable for preconcentration.
- Proteins with a pI less than 4
- Small molecules
How does preconcentration work?
Preconcentration can be achieved by reducing the pH of the activation buffer (the buffer the ligand is dissolved in) to below the pI (isoelectric point) of the ligand. This causes the ligand to be electrostatically attracted to the carboxyl surface, as the ligand will be positively charged, while the carboxyl surface will be negatively charged. This electrostatic attraction overwhelms any steric or hydrophilic stabilization. The ligand will accumulate at the surface until electrostatic neutrality is reached.
Immobilization buffers are generally chosen:
- 0.5 pH units below the pI for pI 3.5 – 5.5
- 1 pH unit below pI for pI 5.5 – 7
- pH 6 for a pI over 7
Using a pH lower than the isoelectric point results in analyte with positive charge, and using a pH higher than the isoelectric point results in analyte with negative charge.
A sensor chip will lose its pre-concentration capacity at a pH lower than 3.
Note: Amine coupling is most efficient at a high pH as activated carboxyl groups react better with uncharged amino groups. Therefore the highest pH compatible with preconcentration should be determined empirically.
Activation buffers are more effective when they have a low ionic strength (less than 20 mM) as this limits charge screening. Buffers with primary amine groups or strong nucleophiles like Tris or sodium azide must be avoided as these will compete with the ligand and lower the immobilization efficiency.
The pH of the activation buffer regulates the rate and amount of the ligand that adsorbs to the sensor chip surface as it changes the surface and ligand charges. The preconcentration rate will also depend on the concentration of the ligand. A lower concentration is preferred as this provides more control over the amount of immobilized ligand. However, dilution of the ligand in the activation buffer may have some undesired effects when the ligand is not concentrated enough. The efficiency of the preconcentration is also proportional to the molecular weight of the ligand.
What are the benefits?
The key benefits of preconcentration include:
- Immobilization of the protein to the sensor chip surface is more efficient
- Far lower concentrations of protein can be injected to get equivalent levels of ligand immobilization
- Preconcentration is independent of the ligand concentration, and works even at concentrations as low as a few ug/ml
Bringing preconcentration into your lab
The first step in preconcentration is to find the optimal preconcentration buffer conditions. This should be done with a carboxyl surface that has not been activated with EDC/NHS to allow many conditions to be tested on a single surface:
- Dilute the ligand to a final concentration of 20-50 µg/mL into the activation buffer with the highest pH
- Decrease pH in steps of 0.5 units, regenerating the surface with 50 mM NaOH to remove the ligand
- If no interaction occurs, the ionic strength or pH should be lowered – increasing the protein concentration is optional but usually not helpful
- Chose the highest pH that gives sufficient immobilization levels
If immobilization is not possible, another immobilization method should be considered.
Once an optimal preconcentration buffer has been determined, add this step into your procedure:
- Wash with activation buffer determined above
- Inject ligand
- Typically protein will bind slower than to an unactivated surface (some of the carboxyl groups have been converted into non-charged groups)
- Yield can be increased by repeated injections of the protein solution
- When working at low pH, inject water to complete the coupling reaction as it will proceed slower at lower pH
- Quench remaining active groups with blocking buffer
- Optional: remove physisorbed proteins with regeneration buffer
- Wash with running buffer
- Switch to suitable running buffer and start interaction experiments
Preconcentration enables researchers to take advantage of electrostatic interactions to increase the localized concentration of ligands on an SPR sensor surface. Adding preconcentration to your SPR procedure will give you the ability to obtain accurate kinetic data, even with a very low ligand concentration.
Nicoya provides an optimized activation buffer for preconcentration within their Amine Coupling Kit. Our application scientists can help you determine the right preconcentration protocol to reduce waste of your precious ligands!
“Preconcentration.” SPRPages http://www.sprpages.nl/immobilization/ligand-pre-concentration
- T. Gedig, Surface Chemistry in SPR Technology. In R. B. M. Schasfoort, A. J. Tudos, Handbook of Surface Plasmon Resonance. Münster, Germany, pp 195-197, 2008.
P. A. van der Merwe, Surface Plasmon Resonance. Oxford, London, pp 12-14, 2001.