Fluorescein-5-maleimide

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.