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.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.