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  3. Fluorescein-5-maleimide

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

Cat. No.: HY-D0098 Purity: 99.05%
COA Handling Instructions

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.

For research use only. We do not sell to patients.

Fluorescein-5-maleimide Chemical Structure

Fluorescein-5-maleimide Chemical Structure

CAS No. : 75350-46-8

Size Price Stock Quantity
5 mg USD 42 In-stock
10 mg USD 66 In-stock
25 mg USD 132 In-stock
50 mg USD 198 In-stock
100 mg   Get quote  
200 mg   Get quote  

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Based on 3 publication(s) in Google Scholar

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Description

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].

In Vitro

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.

Molecular Weight

427.36

Formula

C24H13NO7

CAS No.
Appearance

Solid

Color

Yellow to orange

Emission (Em)

505

Excitation (Ex)

488

SMILES

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

Shipping

Room temperature in continental US; may vary elsewhere.

Storage

-20°C, protect from light

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

Solvent & Solubility
In Vitro: 

DMSO : 50 mg/mL (117.00 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 2.3399 mL 11.6997 mL 23.3995 mL
5 mM 0.4680 mL 2.3399 mL 4.6799 mL
View the Complete Stock Solution Preparation Table

* 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.

  • Molarity Calculator

  • Dilution Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

Mass
=
Concentration
×
Volume
×
Molecular Weight *

Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2

Concentration (start)

C1

×
Volume (start)

V1

=
Concentration (final)

C2

×
Volume (final)

V2

In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

    Solubility: 2.5 mg/mL (5.85 mM); Suspended solution; Need ultrasonic

    This protocol yields a suspended solution of 2.5 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

    Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
  • Protocol 2

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

    Solubility: 2.08 mg/mL (4.87 mM); Suspended solution; Need ultrasonic

    This protocol yields a suspended solution of 2.08 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

    Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

Dosage

mg/kg

Animal weight
(per animal)

g

Dosing volume
(per animal)

μL

Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
%
DMSO +
+
%
Tween-80 +
%
Saline
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration: mg/mL
Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).

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

The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
 If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Purity & Documentation

Purity: 99.05%

References

Complete Stock Solution Preparation Table

* 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.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
DMSO 1 mM 2.3399 mL 11.6997 mL 23.3995 mL 58.4987 mL
5 mM 0.4680 mL 2.3399 mL 4.6799 mL 11.6997 mL
10 mM 0.2340 mL 1.1700 mL 2.3399 mL 5.8499 mL
15 mM 0.1560 mL 0.7800 mL 1.5600 mL 3.8999 mL
20 mM 0.1170 mL 0.5850 mL 1.1700 mL 2.9249 mL
25 mM 0.0936 mL 0.4680 mL 0.9360 mL 2.3399 mL
30 mM 0.0780 mL 0.3900 mL 0.7800 mL 1.9500 mL
40 mM 0.0585 mL 0.2925 mL 0.5850 mL 1.4625 mL
50 mM 0.0468 mL 0.2340 mL 0.4680 mL 1.1700 mL
60 mM 0.0390 mL 0.1950 mL 0.3900 mL 0.9750 mL
80 mM 0.0292 mL 0.1462 mL 0.2925 mL 0.7312 mL
100 mM 0.0234 mL 0.1170 mL 0.2340 mL 0.5850 mL
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Help & FAQs
  • Do most proteins show cross-species activity?

    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

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