Detecting kinase activities from single cell lysate using concentration-enhanced mobility shift assay.

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• High detection sensitivity is crucial for analyzing cellular processes.
• Existing methods for kinase activity detection have limitations in sensitivity and throughput.
• Understanding kinase signaling is key to deciphering cell fate decisions.

Purpose of the Study:

• To develop a microfluidic device for sensitive detection of cellular kinase activities.
• To enable simultaneous concentration and separation of phosphorylated peptides.
• To achieve single-cell resolution for kinase activity profiling.

Main Methods:

• Utilizing electrokinetic preconcentration coupled with mobility shift assays.
• Designing a microfluidic device with nanoliter microwells for cell culture.
• Implementing a novel spacer peptide method to enhance separation resolution.
• Measuring kinase activities from single adherent cells.

Main Results:

• The microfluidic platform achieves high detection sensitivities for kinase activities.
• The device reliably measures kinase activities of single adherent cells.
• Spacer peptides significantly improve separation resolution and concentration factors.
• Multiplexed kinase measurements with single-cell sensitivity are demonstrated.

Conclusions:

• This technology enables precise measurement of kinase activities at the single-cell level.
• The platform facilitates multiplexed kinase profiling from single cell lysates.
• This approach can advance the study of heterogeneous signaling pathways and cell fate determination.