Protocol for reading and imaging live-cell PKA activity using ExRai-AKAR2.

Area of Science:

• Cellular biology
• Biochemistry
• Molecular imaging

Background:

• Fluorescent protein (FP)-based biosensors are crucial for studying signaling pathways in living cells.
• Limited sensitivity of current kinase biosensors hinders high-throughput applications.
• Protein kinase A (PKA) plays a vital role in numerous cellular processes.

Purpose of the Study:

• To develop and validate an ultrasensitive reporter for live-cell PKA activity detection.
• To demonstrate the utility of this reporter in high-throughput modalities.
• To provide a protocol for its application in fluorescence microplate reading and epifluorescence microscopy.

Main Methods:

• Utilized an ultrasensitive excitation-ratiometric PKA activity reporter, ExRai-AKAR2.
• Employed fluorescence microplate reading for high-throughput detection.
• Applied epifluorescence microscopy for detailed imaging.
• Followed established protocols (Mehta et al., 2018; Zhang et al., 2021a).

Main Results:

• ExRai-AKAR2 demonstrated high sensitivity in detecting live-cell PKA activity.
• The reporter is well-suited for high-throughput applications.
• Successful detection of PKA activity was achieved via fluorescence microplate reading and microscopy.

Conclusions:

• ExRai-AKAR2 significantly enhances the sensitivity of kinase activity biosensors.
• This ultrasensitive reporter facilitates reliable high-throughput analysis of PKA signaling dynamics.
• The protocol enables robust live-cell PKA activity measurements in various imaging platforms.