List of SPR Assays
|ACE2||ACE2||Angiotensin I converting enzyme 2||Please inquire|
|BRD4-1||BRD4||Bromodomain containing 4, domain 1||Data sheet|
|BRD4-2||BRD4||Bromodomain containing 4, domain 2||Data sheet|
|cGAS (157-522)||CGAS||Cyclic GMP-AMP synthase, fragment||Data sheet|
|HEMK2||N6AMT1||N-6 adenine-specific DNA methyltransferase 1||Please inquire|
|HIF1a, 2a||HIF1A, EPAS1||Hypoxia-inducible factor 1-alpha, Endothelial PAS domain-containing protein 1||Please inquire|
|NSD2-SET||NSD2||SET domain of NSD2||Data sheet|
|PRMT5/MEP50||PRMT5/ WDR77 complex||Protein arginine methyltransferase 5, WD repeat domain 77||Data sheet|
|SARS-CoV-2 S protein - ACE2 interaction||n.a.||Spike glycoprotein, receptor binding domain; Angiotensin I converting enzyme 2||S protein webpage|
|SOS1||SOS1||SOS Ras/Rac guanine nucleotide exchange factor 1, son of sevenless homolog 1||Data sheet|
Additional Information for SPR Binding Assays
Kinetic profile of an analyte-target binding reaction.
SPR detects changes in the refractive index at the surface of a sensor chip as a result of molecular mass changes of a target upon binding of the analyte. The target is immobilized to the surface of the sensor chip. During the association phase, the analyte flows over the surface, and binding to the target is monitored. The flow then switches to the running buffer, and the dissociation of the analyte from the target is monitored.
Example of a co-factor analysis by SPR.
In this example study, we investigated a substrate competitive inhibitor that binds to its target enzyme, PRMT5/MEP50, only in the presence of SAM or SAM analogs such as MTA and SAH. No dose-dependent responses were observed for analyte binding to apoprotein (left figure). The binding to the MTA-bound target is relatively weak (KD~20µM) with fast kinetics (on/off). The binding affinity increased by ~10-fold for the SAH-bound target (KD~2µM). While the on-rates are similar for the MTA- and SAH-bound conditions, the off-rates are approximately 100X slower. The highest affinity (KD~3nM) and slowest off-rate (100x less than SAH-bound) was observed for analyte binding to the SAM-bound target. Single-cycle kinetics, that do not require a return to baseline in between doses, was used due to the slow off-rate observed for this condition. A slower off-rate indicates longer occupancy of the analyte on the target.
The analyte was tested with 7 concentrations depicted in different colors.
Example of binding affinity determination of an inhibitor to two bromodomains.
The BD1 or BD2 domain of BRD4 were immobilized to the sensor chip surface. The analyte was applied to the chip surface in increasing concentrations approaching saturation.
Right graph: The relative response at equilibrium for each dose was plotted against the analyte concentration to determine the equilibrium dissociation constant, KD. The analyte is 10-times more selective for BD2 than for BD1.
Instruments: Reaction Biology is equipped with two state-of-the-art Biacore 8K units and one 8K+ unit allowing high-throughput screening with 8 channels (4600 compounds per day) with high sensitivity.
Turnaround time: We strive for a 2 week development time for assays with a new target for screening and 2 weeks for the screening with an established assay. Projects are performed on a first-come-first-serve basis.
Screening location: The assay is performed in Malvern, PA, USA.
Sample requirements: The target can be provided by the client, be one of our off-the-shelf proteins, or be custom-made to the requirements of the client and assay. A purity of 90% or more is recommended.
Please see our FAQ for shipping instructions.
Example SPR workflow of a customized analyte-target binding study:
- Sensor chip preparation: Attaching the target to the sensorchip
- Assay validation: Testing the behavior of the target on the sensor chip
- Assay reproducibility: Testing if the optimized conditions are reproducible
- Protein activity and stability on the sensor chip
- Analyte testing
- The binding of the test analyte(s) is measured
- For kinetics/affinity determination either a 10 concentration multi-cycle or 5 concentration single-cycle measurement will be used (dependent upon the off-rates and regeneration conditions)
- For screening studies, a single concentration measurement can be used for analyte ranking