FG 488 DHPE, a lipid-coupled fluorochrome, is used as the fluorophore Oregon Green 488. It monitors the acidification of lipid vesicles with λex/λem=508/534 nm and is also utilized for quantifying Hv1-induced proton translocation with the same excitation/emission wavelengths [1] [2].

FG 488 DHPE exhibits pH-dependent fluorescence emission characteristics [1]. Monitoring acidification in Bulk vesicle assays [1]: 1. Instrument: Jasco FP6500 spectrofluorometer, 37 ℃; excitation at λex=508 nm and emission detection at λem=534 nm. 2. Add 100 μL proteoliposomes (cphospholipid ~60 μM) to 680 μL ATPase buffer with K+-ionophore valinomycin (5 nM) for charge equilibration. 3. Add ATP (1.2 mM) to induce proton pumping. 4. Add 1 mM NaN3 to halt ATP hydrolysis. 5. Add CCCP (carbonyl cyanide 3-chlorophenyl hydrazine, 0.4 μM) to deplete the proton gradient. Conversion into pH-values normalizes fluorescence intensities to those obtained directly after ATP addition. FG 488 DHPE quantifies pH changes induced by the voltage-dependent proton channel Hv1 [2]. Quantification of phospholipid concentrations [2]: 1. Add Perchloric acid (70%, 200 μL) to a sample of unilamellar vesicles containing OG488-DHPE (30 μL). 2. Heat at 220 °C for 60 min to generate inorganic phosphate. 3. Cool to room temperature and add 700 μL of NH4MoO4 (0.45% (w/v)), perchloric acid (12.6% (w/v)), and 700 μL acetic acid (1.7% (w/v)). 4. Obtain a calibration curve for NaH2PO4 concentrations. 5. Incubate samples at 80 °C for 10 min and measure absorption at 820 nm. 6. Calculate phospholipid concentrations using the calibration curve. Proton translocation assay [2]: 1. Instrument: Jasco FP6500 spectrofluorometer, 37 ℃; excitation at λex=508 nm (3 nm band width) and emission detection at λem=534 nm (3 nm band width). 2. Dilute proteoliposomes (POPC/POPG/Chol/OG488-DHPE in 54.5:25:20:0.5 ratio) in buffer A within flux buffer, creating a 14-fold K+-gradient across the membrane. 3. Add valinomycin (13 nM) to induce protonation of OG488-DHPE and quench its fluorescence intensity for active Hv1 channels. 4. Add CCCP (6 nM) to permeabilize vesicles for protons. 5. Plot normalized fluorescence intensity Fnorm as a function of time. Use protein-free vesicles as a control for proton leakage. For experiments with the potential inhibitor 2GBI, dissolve the inhibitor (15 mM) in flux buffer and add (0.5-8.0 μL) to proteoliposomes before valinomycin addition to induce proton translocation.