Coumarins represent small molecular weight blue fluorescent dyes that are UV-excitable. Coumarin dyes are water soluble and permeable across the cell membrane though the permeability may be reduced with the increase in charged moieties. They can be used to provide ideal contrast in multicolor context. As their fluorescence is not as bright, it may be applicable to situations wherein the targets are abundant or for cell tracking purposes to avoid being obscured by autofluorescence. 7-Hydroxycoumarin (also called umbelliferone or umbelliferyls) is highly fluorescent (blue) dye and pH sensitive. As phosphorylation or alkylation of its hydroxyl group quenches fluorescence strongly, it can be used in high-throughput screening for phosphatases. The MUG, MUP and DiFMUP derivatives of coumarin have been used extensively as substrates for detecting enzymatic activity in various contexts (homogenates, cells, solution).
4-Methylumbelliferyl phosphate is useful as a fluorogenic substrate for calmodulin-dependent protein phosphatase (1). It’s also used in kinetic studies of alkaline phosphatase (2). In enzyme immuno assays for the detection of anti-human HIV antibodies, it was shown to be 6-7 times more sensitive than phenolphthalein monophosphate and 8-13 times more sensitive than p-nitrophenyl phosphate as fluorgenic substrate for alkaline phosphatase (3). At pH 10.3, the cleavage product (4-methylumbelliferone) is intensely fluorescent (excitation maximum at 364 nm, emission maximum at 448 nm). At pH 5, the fluorescence is less intense but still easily measurable (excitation maximum shifted to 330 nm) (4).
1. Anthony FA, et al., A spectrofluorimetric assay of calmodulin-dependent protein phosphatase using 4-Methylumbelliferyl phosphate. (1986) Anal. Biochem., 155, 103-107.
2. Fernley HN, Walker PG. Kinetic Behaviour of Calf-Intestinal Alkaline Phosphatase with 4-Methylumbelliferyl Phosphate. (1965) Biochem. J., 97, 95-103.
3. Roberts LM et al., A comparison of the sensitivity and specificity of enzyme immunoassays and time-resolved fluoroimmunoassay. (1991) J. Immunol. Methods 143, 49-56.
4. Meth. Biochem. Anal.,Vol. 17, 254 (1969).
Product Information
MUP (4-Methylumbelliferyl Phosphate) Oligo Labeling
4-methylumbelliferyl phosphate
(4-methyl-2-oxochromen-7-yl) dihydrogen phosphate
-20°C To -70°C
Oligonucleotides are stable in solution at 4°C for up to 2 weeks. Properly reconstituted material stored at -20°C should be stable for at least 6 months. Dried DNA (when kept at -20°C) in a nuclease-free environment should be stable for years.
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