This protocol provides a comprehensive guide for the use of genetically encoded GFP-Aequorin Protein (GAP) indicators to monitor calcium (Ca2+) dynamics within the endoplasmic reticulum (ER). We detail the application of erGAP3, a low-affinity ratiometric sensor that overcomes the challenges of measuring high intraluminal ER calcium concentrations. The methodology covers protein expression assessment, ratiometric imaging procedures, and precise signal calibration. These tools enable stable and quantitative monitoring of ER Ca2+ both dynamically and at rest, providing a vital framework for studying cellular homeostasis and calcium-related signaling pathways.
Rojo-Ruiz et al. (Mon,) studied this question.