Fluorescein-maleimide  (Synonyms: 5-FITC-Maleimide)

Fluorescein-maleimide (5-FITC-Maleimide) is a thiol-reactive fluorescent derivatization reagent and non-specific protein labeling reagent. Fluorescein-maleimide covalently binds to protein thiol groups for protein labeling. Fluorescein-maleimide covalently binds to protein amino and imidazole groups under neutral pH conditions. Fluorescein-maleimide is used for fluorescent labeling of proteins, nucleic acids or other molecules containing one or more thiol groups (Ex/Em = 494/519 nm)^[1].

Guidelines (The following is our recommended experimental protocol. This protocol is for reference only, and specific operations should be adjusted according to your actual needs).
1. Protein Preparation
1) For optimal labeling efficiency, prepare the protein (antibody) at a concentration of 2 mg/mL.
2) The pH of the protein solution is 6.5±0.5. If the pH is lower than 8.0, adjust it with 1 M sodium bicarbonate.
3) If the protein concentration is lower than 2 mg/mL, the labeling efficiency decreases significantly. For optimal labeling efficiency, the recommended final protein concentration ranges from 2 to 10 mg/mL.
4) If the protein contains no free cysteine residues, treat the protein with DTT or TCEP to generate thiol groups. DTT or TCEP converts disulfide bonds into two free thiol groups. If DTT is used, remove free DTT by dialysis or gel filtration before conjugating the maleimide dye to the protein.
2. Dye Preparation
Add anhydrous DMSO to the Fluorescein-maleimide vial to prepare a 10 mM stock solution. Mix thoroughly by pipetting or vortexing.
3. Calculation of Dye Dosage
The amount of Fluorescein-maleimide required for the reaction depends on the amount of protein to be labeled, with an optimal molar ratio of dye to protein of approximately 10:1.
Example: Assuming 500 μL of 2 mg/mL IgG (MW=150,000) is used as the marker protein, and 1 mg of Fluorescein-maleimide is dissolved in 100 μL of DMSO, the required volume of Fluorescein-maleimide is 3.5 μL. The detailed calculation process is as follows:
1) mmol (IgG) = mg/mL (IgG) × mL (IgG)/MW (IgG) = 2 mg/mL × 0.5 mL/150,000 mg/mmol = 6.7×10^-6 mmol
2) mmol (Fluorescein-maleimide) = mmol (IgG) × 10 = 6.7×10^-6 mmol × 10 = 6.7 × 10^-5 mmol
3) μL (Fluorescein-maleimide) = mmol (Fluorescein-maleimide) × MW (Fluorescein-maleimide)/mg/μL (Fluorescein-maleimide) = 6.7 ×10^-5 mmol × 529.52 mg/mmol/0.01 mg/μL = 3.5 μL (Fluorescein-maleimide)
Note: A 10:1 dye-to-protein molar ratio is recommended. If the concentration is too low or too high, adjust the ratio to 5:1, 15:1 or 20:1.
4. Performing the Conjugation Reaction
1) Slowly add an appropriate amount of freshly prepared 10 mg/mL Fluorescein-maleimide to the 0.5 mL protein sample. Gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Do not overmix, as this may cause denaturation and inactivation of the protein sample.
2) Place the reaction tube in the dark, and incubate at room temperature for 60 minutes with gentle inversion every 10-15 minutes.
5. Purification of the Conjugate
The following protocol is an example of purifying the dye-protein conjugate using a Sephadex G-25 column.
1) Prepare the Sephadex G-25 column according to the manufacturer’s instructions.
2) Load the reaction mixture onto the top of the Sephadex G-25 column.
3) Once the sample runs below the surface of the top resin layer, immediately add PBS (pH 7.2-7.4).
4) Add more PBS (pH 7.2-7.4) to complete the column purification. Pool the fractions containing the desired dye-protein conjugate.

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

529.52

C₂₇H₁₉N₃O₇S

2228857-33-6

Solid

Yellow to orange

O=C1OC2(C3=CC=C(NC(NCCN4C(C=CC4=O)=O)=S)C=C13)C5=CC=C(C=C5OC6=CC(O)=CC=C62)O

Room temperature in continental US; may vary elsewhere.

4°C, sealed storage, away from moisture and light

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and 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 (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Taki M, et al. Transglutaminase-mediated N- and C-terminal fluorescein labeling of a protein can support the native activity of the modified protein. Protein Eng Des Sel. 2004 Feb;17(2):119-26.  [Content Brief]

  [2]. Stefanucci A, et al. Fluorescent-labeled bioconjugates of the opioid peptides biphalin and DPDPE incorporating fluorescein-maleimide linkers. Future Med Chem. 2017;9(9):859-869.  [Content Brief]