High-throughput kinase assay based on surface plasmon resonance suitable for native protein substrates

Area of Science:

• Biochemistry
• Assay Development
• Biosensing Technology

Background:

• Kinase assays are crucial for drug discovery and understanding cellular signaling.
• Existing methods like radioisotope assays can be time-consuming and generate radioactive waste.
• There is a need for sensitive, high-throughput, and reliable kinase assay platforms.

Purpose of the Study:

• To develop and validate a novel in vitro high-throughput kinase assay utilizing surface plasmon resonance (SPR).
• To assess the reliability and efficiency of the SPR-based assay compared to traditional radioisotope methods.
• To demonstrate the utility of the SPR assay for various screening applications.

Main Methods:

• In vitro tyrosine phosphorylation was conducted in microtiter plates.
• Substrates were captured on an SPR sensor chip using antibodies.
• Phosphotyrosine (pTyr) detection was performed using an anti-pTyr antibody on a Biacore A100 SPR biosensor.
• Assay performance was evaluated by measuring Fyn tyrosine kinase phosphorylation of substrate proteins.

Main Results:

• The high-throughput SPR (HTP-SPR) assay demonstrated high-quality data sampling and processed up to 1000 samples per day.
• Results obtained using the HTP-SPR method closely matched those from a standard radioisotope assay.
• The reliability of the HTP-SPR method was confirmed through comparative analysis with Fyn tyrosine kinase.

Conclusions:

• The developed HTP-SPR assay is a sensitive, reliable, and efficient tool for kinase research.
• This SPR-based method is suitable for high-throughput screening of kinase activity, substrate profiling, and inhibitor screening.
• The HTP-SPR assay presents a powerful alternative to conventional methods, offering significant advantages in throughput and data quality.