Analysis the Kinetics/Affinity of HMGB1–TLR4/MD-2 Interactions by Surface Plasmon Resonance (SPR) (CAT#: STEM-MB-0542-CJ)

Surface Plasmon Resonance (SPR) is an optical technique used to measure molecular interactions in real time. SPR can occur when plane-polarized light hits a metal film under total internal reflection conditions. SPR signal is directly dependent on the refractive index of the medium on the sensor chip. The binding of biomolecules results in changes in the refractive index on the sensor surface. In an SPR experiment, one molecule (the Ligand) is immobilized on a sensor chip and binding to a second molecule (the Analyte) is measured under flow. Response is measured in resonance units (RU) and is proportional to the mass on the surface, and for any given interactant, the response is proportional to the number of molecules bound to the surface. Response is recorded and displayed as a sensogram in real time. SPR experiments can be used to measure kinetic binding constants (ka, kd) and equilibrium binding constants (affinity, Ka = 1/Kd).

Pharmacology; Medical

The instrument capable of automated or manual surface preparation, fully automated injection of test samples in well, and output of SPR data as proprietary datafiles for evaluation with software.
1. Receptor Immobilisation (Surface Preparation): This stage is required to coat the biosensor surface with the target receptor (protein) to which the test compound (ligand) will bind. A variety of different immobilization / capture strategies are available, depending on the nature of the receptor. Usually, a control receptor (protein) to which the test compound won't bind, is also coated onto a reference surface to act as a 'blank'.
2. Ligand Binding and Dissociation: (Sample Injection and Wash-off Intervals) After establishing a stable baseline with buffer alone flowing over the sensor surface, sample is passed over the receptor surface for binding to occur. This period defines the binding association rate. After a defined interval, sample flow ceases and flow of buffer alone is resumed. This allows the bound ligand to wash off, defining the dissociation rate. The duration of the ligand binding and ligand dissociation intervals needs to be sufficient to measure binding kinetics but should be as short as practical to conserve reagents and increase sequential sample throughput.
3. Surface Regeneration (Bound Ligand Removal): Samples are passed over the sensor surface sequentially. For low-affinity ligands, dissociation can be complete (back to initial baseline level) within a few minutes, allowing the next sample to be injected onto a 'clean' sensor surface after a short delay. For high affinity ligands, complete dissociation back to baseline can take hours, but only a few minutes of dissociation rate data are required for analysis. To remove all bound ligand from the sensor surface before the next sample is injected, a 'regeneration' step can be used. This process applies a mild pH change, ionic strength change, or some other biophysical process to rapidly dissociate the ligand from the receptor surface without damaging or denaturing the receptor protein. After returning to baseline with standard buffer flow, the next sample can be injected.
4. Data Analysis: Automated data analysis is performed using proprietary evaluation software.
5. Data Output.

• Sample: Complex mixtures (such as: cell culture supernatants, cell extracts, purified interactants); Small molecules; Peptides; Proteins; Antibody; Nucleic acids; Lipids; Virus-like particles (VLPs); Hormones/Cytokines; Adeno-associated viruses (AAV)
• Equipment: Biacore™ Surface Plasmon Resonance (SPR)
• Running buffer; DMSO; Detergent
• (Optional) 96-well and 384-well reagent plates and foil

1. One of the samples to be tested must be a protein and both samples must be of high purity.
2. Minimum sample sizes available: proteins (50-100μg), small molecules (20μM/200μL), proteins and peptides need to be sent at low temperature.
3. If use DMSO, make sure all equipment (pipette tips, microplates, vials, filters, and so on) are compatible with organic solvents. Do not use polystyrene products with DMSO.