Naph-EA-mal (Thiol-green 1) is a highly efficient and quick-responding thiol fluorescence probe specifically designed for protein labeling and bioimaging applications. This compound offers a rapid detection method and exhibits ultrafast turn-on fluorescence upon interaction with thiols. Naph-EA-mal (Thiol-green 1) enables the detection of thiols in live cells, facilitates the labeling of protein thiols, enables quantification of total thiol concentration in cell lysate, and allows for the determination of reversible protein thiol oxidation in fixed cells. It possesses an excitation wavelength of 488 nM and an emission wavelength of 540 nM.

In a study on imaging thiols within living cells, Hep G2 cells were exposed to Naph-EA-mal (Thiol-green 1) at a concentration of 1 μM for 5 minutes, resulting in strong green fluorescence. However, preincubation of cells with NEM (a thiol-blocking agent; 100 μM) led to notably weaker fluorescence, indicating the compound's sensitivity to intracellular thiol levels[1]. In separate experiments aimed at monitoring thiol-disulfide balance in fixed cells, Hep G2 cells displayed intense fluorescence upon treatment with Naph-EA-mal (10 μM), attributable to the high incidence of reduced thiols. This fluorescence significantly diminished following diamide exposure, which promotes oxidation. Subsequently, cells oxidized with diamide (200 μM for 30 minutes) were fixed with 70% ethanol, treated with NEM (100 μM) to block remaining free thiols, and then exposed to TCEP (5 mM) before being reintroduced to Naph-EA-mal (10 μM). This protocol reinstated the green fluorescent signal, underscoring the methodology's utility in assessing thiol-disulfide dynamics within cells[1].