Assay development and experimental optimization are essential for obtaining high-quality SPR data. Selecting the most suitable design and optimizing the experimental conditions is of utmost importance for maintaining assay accuracy and reproducibility. This is also one of the most time-consuming steps for SPR researchers due to the low throughput of conventional SPR devices.
The ProteOn™ XPR36 protein interaction array system's novel 6 x 6 fluidics afford the researcher the flexibility to run many different types of experiments on a single platform at a higher-throughput than traditional binding assays. This flexibility in assay design allows for streamlining of large-scale sample processing and optimization.
Related Topics: Label-Free Validation and Characterization and Large and Small Molecule Screening by SPR
The unique 6 x 6 interaction array of the ProteOn system can be used to detect binding between up to six targets and six analytes at the same time, greatly enhancing experimental throughput and the versatility of SPR technology for a wide range of applications. An example of this 6 x 1 kinetic screening approach is seen through the investigation of the model system, TEM-BLIP, where 5 mutant TEM1 proteins are immobilized to the sensor chip surface. A single injection of a concentration series of the BLIP wild type analyte yields robust kinetic rate constants that can be used to model future experiments. (Bronner et al. 2006a, Bronner et al. 2009).
Fig. 1. Working model depicting the binding domain interactions between TEM1 and BLIP proteins.
Kinetic constants for the interactions between mutants of TEM1 and wild-type BLIP. The equilibrium dissociation constant, KD, was calculated from kd/ka.
Relevant webinars for assay development:
Kinetic Screening of an scFv Antibody Fragment Library Using the ProteOn XPR36 Interaction Array SystemPresented by Olan Dolezal, PhD (CSIRO Molecular and Health Technologies, Australia)Click to download recorded webinar: https://biorad.box.net/shared/ra1bne7z9c
A Calcium-Dependent Immunocapture Strategy for Enhanced-Throughput SPRPresented by John Kulman, PhD (Puget Sound Blood Center, USA)Click to download recorded webinar: https://biorad.box.net/shared/j00jbuzdet
The Study of Small Molecule-Protein Kinase Interactions using Multiplexed SPRPresented by Tsafrir Bravman, PhD (Bio-Rad Laboratories)Click to download recorded webinar: https://biorad.box.net/shared/p0hrnbgxaria4lyuqfkz
Enhancing Throughput of the ProteOn Biosensor in Antibody Screening ApplicationsPresented by Kevin Lindquist (Pfizer Rinat, USA)Click to download recorded webinar: https://biorad.box.com/s/meykvrgruuff29oqdrod
The novel microfluidics of the ProteOn system uniquely enable researchers to quickly determine ideal experimental conditions for their interaction of interest. XPR™ technology allows the user to investigate up to six conditions of the target followed by a single injection of up to six conditions of the analyte. The 1 x 1 full optimization configuration of the ProteOn enables rapid determination of the surface density of the target, buffer, or regeneration conditions for the experiment. The combination of different varying target conditions interacted with different analyte concentrations results in a large matrix of optimization conditions. Combing through these optimization conditions sequentially can be difficult and time-consuming; however, by using the ProteOn system, a researcher can complete an entire optimization experiment in two injections (Bronner et al. 2006b).
pH dependence of TEM1 immobilization. TEM1 protein was immobilized in ProteOn acetate buffer, pH 3.0–5.0. Ligand density was determined from the average SPR response of the six interaction spots along each ligand channel.
Fig. 2. The dependence of the BLIP-F142A analyte response, TEM1 ligand density, and TEM1 ligand activity (%Rmax) on the pH of the immobilization buffer. Scales adjusted to align the response of each parameter to the same plot.
Webinars addressing experimental optimization using the ProteOn XPR36 system:
An Introduction to the ProteOn XPR36 Surface Plasmon Resonance (SPR) Interaction SystemPresented by Tsafrir Bravman, PhD (Bio-Rad Laboratories)Click to download recorded webinar: https://biorad.box.net/shared/mhfscze1oz
Bronner V. et al. (2006a). Analysis of multiple protein-protein interactions using the ProteOn™ XPR36 protein interaction array system. Bulletin 5368.This tech note describes the investigation of the relative contributions of specific protein substructures and residues to the binding interface between TEM1 β-lactamase (TEM1) and the β-lactamase inhibitor protein (BLIP) (Figure 1) (Albeck and Schreiber 1999). TEM1. Mutated TEM1 residues were used to analyze the consequences of mutations on the binding energetics of the protein interface. Central to this analysis was the fast and accurate "one-shot kinetics" capability of the ProteOn XPR36 protein interaction array system.
Bronner V et al. (2006b). Rapid and efficient determination of kinetic rate constants using the ProteOn XPR36 protein interaction array system. Bio-Rad Bulletin 3172.The tech note employs the ProteOn system to determine the kinetics of the interaction between interleukin-2 (IL2) and anti-IL2 antibody. The experiment was performed using the One-shot Kinetics™ approach to monitor the interaction between multiple concentrations of the analyte IL2 and the ligand anti-IL2 antibody immobilized with multiple conditions in a single analyte injection. The 6 x 6 interaction array was used to generate 36 sensorgrams simultaneously. It not only increased throughput but also provided novel referencing options.
Bronner V et al. (2009). Rapid screening and selection of optimal antibody capturing agents using the ProteOn XPR36 protein interaction array system. Bio-Rad Bulletin 5820.The tech note describes how the One-shot Kinetics approach was used to rapidly screen the binding of four antibody capturing agents and seven types of antibody targets. The selection of antibody-binding proteins that provide the optimal binding characteristics for the capture of each antibody type was achieved rapidly in the ProteOn system. The One-shot Kinetics approach allows for the analysis of multiple experimental conditions in a single experiment.
General ProteOn Literature
Large Molecule Protein-Interaction Analysis
Small Molecule Analysis
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