Coelenterazine h

Coelenterazine h (2-Deoxycoelenterazine), a coelenterazine derivative, is a luminescent substrate for RLuc8. Coelenterazine h is more sensitive to Ca²⁺, thus providing a valuable tool for measuring small changes in Ca²⁺ concentrations (Ex/Em = 437/466 nm)^[1][2][3][4]. In Vitro:1. Solution preparation^[4]
1.1 Preparation of stock solution
Solvent: methanol or ethanol
Concentration: 10 mM (optimized according to the experiment).
1.2 Preparation of working solution
Dilute to 10 µM with PBS or cell culture medium (optimized according to the experiment).
Note: The stock solution and working solution should be prepared and used immediately. Keep it away from light.

2. Cell staining
2.1 Hela cells are transfected with BRAC or G5A.
2.2 Add 10 µM Coelenterazine h into the cell plate.
2.3 Incubate the dye-loading plate in a cell incubator for 1-4 h (optimized according to the experiment).
2.4 Fluorescence microscopy detection (Ex/Em = 437/466 nm).
Coelenterazine h (1-10 μM) can be used as the luminescent substrate for RLuc8^[4].
In the measurements of Ca²⁺ binding kinetics of BRAC, emission intensity of Venus (530 nm) from BRAC are monitored at 1 kHz just after rapid mixing of 5 nM BRAC protein with 20 μM coelenterazine-h in various concentration of Ca²⁺ buffer^[4].