MUP [4-Methylumbelliferyl phosphate, free acid] *CAS 3368-04-5*

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Chemical structure for MUP [4-Methylumbelliferyl phosphate, free acid] *CAS 3368-04-5*
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Ex/Em (nm)360/449
CAS #3368-04-5
Storage Freeze (<-15 °C)
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Category Enzyme Detection
Related Protein Phosphatase
A fluorogenic substrate for phosphatases. It is widely used for detecting phosphatases in solution. However this phosphatase substrate is not well suited for living cell or continuous assays since MU (4-methylumbelliferone), the enzymatic product, which only develops maximum fluorescence at pH value of >10. Thus it is also difficult to use MUP for the detection of phosphatases that have acidic optimal pH range such as acid phosphatases. AAT Bioquest is pleased to offer MUP Plus that is developed to address this pH limitation associated with MUP substrates.

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of MUP [4-Methylumbelliferyl phosphate, free acid] *CAS 3368-04-5* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

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Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =

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This protocol only provides a guideline, and should be modified according to your specific needs.

1. Prepare working solution:


1.1    Prepare a 2 to 10 mM stock solution in an appreciate solvent (see Table). The stock solution should be used promptly. Any unused solution need to be aliquoted and frozen at < -20 oC.

Note 1: Do not use DMSO, ETOH or METH to make stock solution of SunRed™ Phosphate (cat#11629) since it significantly increases assay background.

Note 2: Avoid repeated freeze-thaw cycles, and protect from light.

1.2    Prepare a 2X Phosphate working solution:  On the day of the experiment, either dissolve the substrate in an appreciate solvent or thaw an aliquot of the stock solution (from Step 1.1) at room temperature. Prepare a 2X working solution of 10 to 50 µM in 100 mM Tris buffer or buffer of your choice, pH 8 to 9 (not phosphate buffer).



2. Run phosphatase assay in supernatants:


2.1    Add 50 µL of 2X Phosphate working solution (from Step 1.2) into each well of the phosphatase standard, blank control, and test samples to make the total phosphatase assay volume of 100 µL/well.

Note: For a 384-well plate, add 25 µL of sample and 25 µL of 2X Phosphate working solution into each well.


2.2    Incubate the reaction for 30 to 120 minutes at the desired temperature, protected from light.


2.3    Monitor the fluorescence increase at an appropriate filter sets (See table) with a fluorescence plate reader.


2.4    The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the phosphatase reactions.

References & Citations

Application of intracellular alkaline phosphatase activity measurement in detection of neutrophil adherence in vitro
Authors: Bednarska K, Klink M, Sulowska Z.
Journal: Mediators Inflamm (2006): 19307

Evaluation of an alternative method for the enumeration and confirmation of Clostridium perfringens from treated and untreated sewages
Authors: Wohlsen T, Bayliss J, Gray B, Bates J, Katouli M.
Journal: Lett Appl Microbiol (2006): 438

Fluorometric cell-ELISA for quantifying rabies infection and heparin inhibition
Authors: Rincon V, Corredor A, Martinez-Gutierrez M, Castellanos JE.
Journal: J Virol Methods (2005): 33

Purification and partial characterization of an acid phosphatase from Spirodela oligorrhiza and its affinity for selected organophosphate pesticides
Authors: Hoehamer CF, Mazur CS, Wolfe NL.
Journal: J Agric Food Chem (2005): 90

Relative movement and soil fixation of soluble organic and inorganic phosphorus
Authors: Anderson BH, Magdoff FR.
Journal: J Environ Qual (2005): 2228

Crystal structure of a covalent intermediate of endogenous human arylsulfatase A
Authors: Chruszcz M, Laidler P, Monkiewicz M, Ortlund E, Lebioda L, Lewinski K.
Journal: J Inorg Biochem (2003): 386

Development of a rapid 1-h fluorescence-based cytotoxicity assay for Listeria species
Authors: Shroyer ML, Bhunia AK.
Journal: J Microbiol Methods (2003): 35

Enzymatic differentiation of Candida parapsilosis from other Candida spp. in a membrane filtration test
Authors: Bauters TG, Peleman R, Dhont M, Vanhaesebrouck P, Nelis HJ.
Journal: J Microbiol Methods (2003): 11

A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration
Authors: Bouaicha N, Maatouk I, Vincent G, Levi Y.
Journal: Food Chem Toxicol (2002): 1677

LVV-hemorphin-4 modulates Ca2+/calmodulin-dependent pathways in the immune system by the same mechanism as in the brain
Authors: Barkhudaryan N, Gambarov S, Gyulbayazyan T, Nahapetyan K.
Journal: J Mol Neurosci (2002): 203

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