Surface Plasmon Resonance

Biorad ProteOn XPR36

Surface Plasmon Resonance (SPR) measurements measures biomolecular interactions in a real-time and label-free. SPR experiments can characterize the following biomolecular interaction aspects:

  1. specificity of interaction (yes/no)
  2. rate of association (ka) ("recognition rate")
  3. dissociation rate (kd) (stability rate of complex)
  4. affinity constant (KD) (ie. strength of interaction: Kd = kd/ka)
  5. thermodynamics (SPR measurements completed at different temperatures).

The Biorad Proteon XPR36 instrument has a 6 x6 parallel flow channels (6 horizontal and 6 vertical flow channels) configuration. This flow design allows for fast experimental optimization, and high throughput screening (up to 36 different ligands immobilized on one sensor chip).

The system is fully automated, so experiments can be programmed and instrument left unattended.

Advantages

  • Real-time biomolecular interactions
  • Small sample volume requirements
  • Label free technique
  • Different sensor chips available to immobilize variety of tagged biomolecules (His-tags, GST-tags, biotinylated, etc.)
  • Recovery of surface bound samples for MS analysis

Specifications

Refractive Index Range 1.33 to 1.37
Number of Flow Channels 6 Horizontal, 6 Vertical
Number of Interaction Spots 36 (0.2025 mm2)
Number of Interspot References 42
Detection Temperature Range 15-40 °C (max. 10 °C below ambient temperature)
Baseline Noise < 1 RU (< 20 kRu); < 1.5 RU (20-40 kRU)
Baseline Drift < 1 RU/min (15-40 °C)
Flow Rate 25-200 μL/min
Sample Injection Volume 25-449 μL
Minimum Sample Volume 93 μL (35+25+8 μL for system+vial dead volume)
AutoSampler 2 x 96-well plates (8 x 12 configuration)
Buffer Valves Switch between 2 different running buffers
Online Degasser For Buffer system only
Detection Limits (Typical Values*)
Concentration 10-3 to 10-10 M
Association Rate Constant (ka) 103 to 107 M-1 s-1
Dissociation Rate Constant (kd) 10-5 to 10-1 s-1
Affinity (kinetic) (ka/kd) 104 to 1011 M-1
Affinity (steady state) 104 to 109 M-1
MW Limit* 201 Da
*Many experimental factors can expand or contract these working ranges and limits. Typical values achieved are listed.

More Information

Figure 1. General overview of a “reference” subtracted sensorgram. (1) A “ligand” biomolecule is immobilized onto a sensor chip surface using different capture techniques (His-tag, Biotin-tag, antibody capture, etc.) or coupling chemistries (amine, thiol, aldehyde coupling, etc.). (2) A second “analyte” biomolecule is injected over the ligand surface, and an interaction with the ligand results in an increase in response (ie. Change in refractive index at surface). (3) Given enough time, the ligand/analyte interaction could reach a steady-state equilibrium position. (4) The analyte solution is replaced with running buffer, and the dissociation of the analyte biomolecule is monitored. (5) A regeneration step is sometimes required to break-up strong ligand/analyte complexes, allowing the sensor surface to be reused.

Why are biomolecular interaction kinetics important? Isn’t a KD good enough?

The affinity constant, KD, is generally used to assess the strength of binding in a biochemical interaction. However, the kinetics of the interaction can reveal significant and meaningful information that cannot be obtained by other methods that evaluate only KD. Interactions with the same affinity can have different orders of magnitude association and dissociation rate constants (see Figure 2 below). For example, SPR kinetic measurements are used extensively in the pharmaceutical industry in order to evaluate potential drug candidates. An antibody or small molecule that may have a high affinity (low KD) but a high kd (fast dissociation) can be easily replaced in vivo, and would likely not a good drug candidate.

Figure 2. SPR sensorgrams of showing the changes in binding kinetics (ka, kd) profiles for different biomolecular interactions having the same affinity (Kd = 5.0 nM) using the same concentration of analyte (50 nM) and same affinity

Guide for Starting SPR Protocols

  1. Will SPR experiments work for your system?

    To determine whether the SPR system is right for your interaction of interest, a simple calculation can determine what kind of theoretical response maximum (Rmax) is possible with your interaction: R_max=(?MW?_A/?MW?_L ) R_L n

    Where MWA MWL are the molecular weights of analyte and ligand, RL is the amount of surface immobilized ligand, and n is the interaction stoichiometry. Note that the theoretical Rmax is possible only if 100% of the active binding sites are available.
  2. Pre-Experiment Homework

    Before immediately jumping into the lab, one should do a little homework and ask yourself:

    • What kind of information do I want to obtain? Do I want simple yes/no binding? Affinity constants? Rate constants?
    • Are there any similar SPR results in the literature? Do I have other experimental information pertaining to the binding interaction?
    • What kind of purity are my biomolecules? (ideally they should be >95% pure)
    • Which biomolecule should I immobilize?
    • Can I take advantage of any tags?
    • How will I immobilize the ligand onto the surface (covalent immobilization via amine coupling vs. capture via His-tag, GST-tags, etc.)?
    • What kind of sensor chip should I use?
    • Are there any other molecules in the ligand solution that may prevent coupling? Detergents, stabilizing proteins, etc.?
    • What are my controls?
    • What supporting experiments do I have to compliment the SPR results?

Links