Flubida-2 is a cell-permeable dye hydrolyzed by intracellular esterases to Fubi-2 (post-hydrolysis, Ex=492 nm, Em=517 nm). By directing the probe to the location of biotin-fusion proteins, Flubida-2 is useful for measuring pH at specific sites within organelles.

Flubida-2 Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). Begin by dissolving Flubida-2 in DMSO at a concentration of approximately 2 mM. Next, prepare the stock solution by mixing it 1:1 with 20% (w/v in DMSO) Pluronic F-127 (Molecular Probes), and dilute to the desired final concentration of 2-4 μM using serum-free DMEM. After transfection with either AV-KDEL or ST-AV DNA, HeLa cells are washed once with serum-free DMEM and then incubated with 2-4 μM Flubida-2 for 3-5 hours, or overnight for 10-15 hours, 30 to 48 hours post-transfection. Subsequently, chase the labeled cells with normal growth medium for at least 2 hours to facilitate the removal of excess dye-biotin from the cytosol. The robust avidin-biotin interaction ensures stable and specific avidin-Flubida binding that withstands washing. Biotin starvation of cells is not required before Flubida loading, as the staining remains bright and stable.