A fluorescent marker for autophagic vacuoles
Monodansylcadaverine (MDC) is a fluorescent marker for autophagic vacuoles.1,2 It is an autofluorescent substance incorporated into multilamellar bodies by both an ion trapping mechanism and interaction with membrane lipids, exhibiting a Stokes shift and increased relative fluorescence in hydrophobic environments.1 MDC selectively accumulates in PaTu 8902 subcellular fractions containing the lysosomal enzymes acid phosphatase and cathepsin D, but not those containing the rough and smooth endoplasmic reticulum markers TRAM and cytochrome P450, respectively.2
1.Niemann, A., Takatsuki, A., and Els?sser, H.P.The lysosomotropic agent monodansylcadaverine also acts as a solvent polarity probeJ. Histochem. Cytochem.48(2)251-258(2000) 2.Biederbick, A., Kern, H.F., and Els?sser, H.P.Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuolesEur. J. Cell Biol.66(1)3-14(1995)
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Kinase experiment: |
To determine the time course of transglutamination, thymosin β4 (120 μM) is incubated with Dansylcadaverine (5 mM) in 70 μL buffer consisting of 10 mM Tris-HCl, pH 7.4, 15 mM CaCl2, 3 mM DTT. The reaction is started by addition of 0.1 U transglutaminase. Immediately after addition of the enzyme (t=0) and at indicated times,10 μL are taken from the mixture, diluted in 490 μL 0.1% TFA to stop the reaction and analyzed by HPLC[1]. |
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Cell experiment: |
MCF7 cells (2.4×104) are seeded into 35 mm plates. After 24 h incubation, CuO NPs are added with an increasing concentration in the presence or absence of 3-Methyladenine (3-MA) for different time periods . The cells are then incubated with 50 mM Dansylcadaverine (MDC) at 37°C for 15 min and washed with 1×PBS three times with 5 min interval. Finally, the cells are observed under a fluorescence microscope[2]. |
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参考文献: [1]. Huff T, et al. Thymosin beta(4) serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescentdansylcadaverine does not abolish interaction with G-actin. FEBS Lett. 1999 Dec 24;464(1-2):14-20. |
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