Fluorescein-5-maleimide  (Synonyms: N-(5-Fluoresceinyl)maleimide)

Fluorescein-5-maleimide (N-(5-Fluoresceinyl)maleimide) is a fluorescent dye. Fluorescein-5-maleimide can be used to detect the redox state of thiols in eukaryotic cells. Fluorescein-5-maleimide can label peptides and is used to detect negatively charged nanoparticles. Fluorescein-5-maleimide can also label actin to explore its interaction with cardiac myosin-binding protein C (cMyBP-C), which helps in developing small molecule modulators for heart failure. Fluorescein-5-maleimide can screen mutant proteins that contain cysteine residues. The excitation wavelength of Fluorescein-5-maleimide is 494 nm, and the emission wavelength is 519 nm^[1][2][3][4].

Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.)
Protein Labeling Experiment Plan Protein preparation: Dissolve the protein in an appropriate buffer (e.g. 20 mM sodium phosphate, 150 mM NaCl, pH 7.2). Add 5-10 mM EDTA to prevent the formation of disulfide bonds catalyzed by metals.
2. Reversion: If necessary, treat with 20 mM DTT (dithiothreitol) for 30 minutes to reduce the disulfide bonds, and then remove the DTT using a filtration column.
3. Dye Preparation: Dissolve fluorescein-5-maleimide (which can be purchased from suppliers such as AAT Bioquest) in anhydrous DMF (N,N-dimethylformamide) or DMSO (dimethyl sulfoxide) to prepare a 10 mM solution.
4. Coupling: Add 10 to 25 times the molar excess of the dye to the protein solution.
5. Incubation: Incubate at room temperature for 2 hours, or incubate at 4°C in the dark overnight.
6. Purification: Use a desalination column, dialysis, or purification column (such as Sephadex G-25) to remove the unreacted dyes.
Important Notes
pH value: Maintain the pH value between 6.5 and 7.5. A higher pH value will increase the reaction rate, but it may lead to non-specific labeling of amino groups.
2. Stability: The malononitrile reagent is sensitive to moisture; please store it under an inert gas atmosphere.
3. Excitation/Emission Wavelength: Approximately 494/519 nm.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

427.36

C₂₄H₁₃NO₇

75350-46-8

Solid

Yellow to orange

505

488

O=C(C=C1)N(C2=CC3=C(C4(C5=C(OC6=C4C=CC(O)=C6)C=C(O)C=C5)OC3=O)C=C2)C1=O

Room temperature in continental US; may vary elsewhere.

-20°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Wadley AJ, et al. Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: An optimised protocol for digital flow cytometers. MethodsX. 2018 Nov 1;5:1473-1483.  [Content Brief]

  [2]. Akinloye O, et al. Peptide-based fluorescence biosensors for detection/measurement of nanoparticles. Anal Bioanal Chem. 2017 Feb;409(4):903-915.  [Content Brief]

  [3]. Bunch TA, et al. Drug discovery for heart failure targeting myosin-binding protein C. J Biol Chem. 2023 Dec;299(12):105369.  [Content Brief]

  [4]. McLachlin DT, et al. A method of screening for mutant proteins containing cysteine residues using fluorescein-5-maleimide. Protein Expr Purif. 1996 May;7(3):275-80.  [Content Brief]