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].

Preparation of Fluorescein-5-maleimide Fluorescent Probe^[1]
1.1 Preparation of storage solution
Prepare 1 mM Fluorescein-5-maleimide storage solution using d-PBS.
Note: 1) Store in the dark.
Fluorescein-5-maleimide for detecting the redox status of thiols in cells
1) Prepare PBMCs from venous blood using Ficoll Paque density centrifugation and wash them in d-PBS.
2) Wash PBMCs (10⁶cells in 100 μ L) labeled with anti human CD3-APC.
3) Stain with 0.1 μ M Fluorescein-5-maleimide and analyze by flow cytometry.

Preparation of Fluorescein-5-maleimide Fluorescent Probe^[2]
1.1 Preparation of working fluid
Prepare 50 mM Fluorescein-5-maleimide with DMSO.
Fluorescein-5-maleimide fluorescent probe for detecting nanoparticles
1) Add the working solution (probe: peptide) in a 5:1 molar ratio to a peptide solution (4.06 mM) containing 20.3 mM N - (2-hydroxyethyl) piperazine-N '- ethanesulfonic acid KOH, 50 mM KCl, and 2 mM tris (2-carboxyethyl) phosphine buffer for peptide labeling.
2) Incubate the mixture (final volume of 125 μ L) in the dark at room temperature for 2 hours and gently shake.
3) Add excess DDT (20 mM) to quench the reaction.
4) Further purify the pure labeled peptide mixture by precipitation with acetone (1 mL).
5) Add 5 μ L of fluorescent probe to 15 μ L of nanoparticles and 130 μ L of buffer solution to detect their interaction.
6) Data analysis: Detecting nanoparticles-peptide interactions through changes in fluorescence intensity of labeled peptides and/or differential light scattering.

Fluorescein-5-maleimide is used to detect protein interactions (actin and cardiac myosin binding protein C (cMyBP-C))^[3]
1) 50 μ M G-actin was polymerized by adding 3 M KCl (final concentration of 100 mM) and 0.5 M MgCl2 (final concentration of 2 mM) under the conditions of 20 mM Tris pH 7.5, 0.2 mM CaCl2, and 0.2 mM ATP, and incubated at 23 ° C for 1 hour.
2) Add Fluorescein-5-maleimide (final concentration of 1 mM) and label at 23 ° C for 1 hour.
3) Add a five fold molar excess of DTT to stop labeling.
4) Incubate 0.25 μ M Fluorescein-5-maleimide actin and 0-20 μ M TMR-cC0-C2 (N-terminal fragment of cMyBP-C) together.
5) Data analysis (FLT-FRET assay): Firstly, excite Fluorescein-5-maleimide and detect its FLT to measure the changes in fluorescence resonance energy transfer (FRET) from Fluorescein-5-maleimide (donor) to TMR (acceptor), with a decrease in the FLT (and an increase in FRET) of Fluorescein-5-maleimide-actin indicating binding.

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]