Fluorescein-5-maleimide (solution)

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Fluorescein-5-maleimide for detecting the redox status of thiols in cells
1.1 Prepare PBMCs from venous blood using Ficoll Paque density centrifugation and wash them in d-PBS.
1.2 Wash PBMCs (10⁶cells in 100 μ L) labeled with anti human CD3-APC.
1.3 Stain with 0.1 μ M Fluorescein-5-maleimide and analyze by flow cytometry.
2. Preparation of Fluorescein-5-maleimide Fluorescent Probe^[2]
2.1 Preparation of working fluid
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.
2.2 Incubate the mixture (final volume of 125 μ L) in the dark at room temperature for 2 hours and gently shake.
2.3 Add excess DDT (20 mM) to quench the reaction.
2.4 Further purify the pure labeled peptide mixture by precipitation with acetone (1 mL).
2.5 Add 5 μ L of fluorescent probe to 15 μ L of nanoparticles and 130 μ L of buffer solution to detect their interaction.
2.6 Data analysis: Detecting nanoparticles-peptide interactions through changes in fluorescence intensity of labeled peptides and/or differential light scattering.
3. Fluorescein-5-maleimide is used to detect protein interactions (actin and cardiac myosin binding protein C (cMyBP-C))^[3]
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.
3.2 Add Fluorescein-5-maleimide (final concentration of 1 mM) and label at 23 ° C for 1 hour.
3.3 Add a five fold molar excess of DTT to stop labeling.
3.4 Incubate 0.25 μ M Fluorescein-5-maleimide actin and 0-20 μ M TMR-cC0-C2 (N-terminal fragment of cMyBP-C) together.
3.5 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. Further protocols information, click here.