5-FAM  (Synonyms: 5-Carboxyfluorescein)

5-FAM (5-Carboxyfluorescein) is a green fluorescent reagent used for in situ labeling peptides, proteins and nucleotides. 5-FAM is a single isomer with Ex/Em of 490 nm/520 nm^[1].

Protocol
1.Protein Preparetion
1) In order to obtain the best labeling effect, please prepare the protein (antibody) concentration as 2 mg/mL.
2) The pH value of protein solution shall be 8.5±0.5. If the pH is lower than 8.0, 1 M sodium bicarbonate shall be used for adjustment.
3) If the protein concentration is lower than 2 mg/mL, the labeling efficiency will be greatly reduced. In order to obtain the best labeling efficiency, it is recommended that the final protein concentration range is 2-10 mg/mL.
4) The protein must be in the buffer without primary amine (such as Tris or glycine) and ammonium ion, otherwise the labeling efficiency will be affected.
2.Dye Preparation
Add anhydrous DMSO into the vial of 5-FAM to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of 5-FAM required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of 5-FAM SE to protein is about 10.
Example: assuming the required marker protein is 500 μL 2 mg/mL IgG (MW=150,000), use 100 μL DMSO dissolve 1 mg 5-FAM, the required 5-FAM volume is 2.52 μL, and 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 (5-FAM) = mmol (IgG) × 10 = 6.7×10^-6 mmol×10 = 6.7 × 10^-5 mmol
3) μL (5-FAM) = mmol (5-FAM) ×MW (5-FAM)/mg/μL (5-FAM) = 6.7 ×10^-5 mmol × 376.32 mg/mmol / 0.01 mg/μL = 2.52 μL (5-FAM)
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL 5-FAM is slowly added to 0.5 mL protein sample
In solution, gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Don'tmix well to prevent protein samples from denaturation and inactivation.
2) The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency.
5.Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a SepHadex G-25 column.
1) Prepare SepHadex G-25 column according to the manufacture instruction.
2) Load the reaction mixture (From "Run conjugation reaction") to the top of the SepHadex G-25 column.
3) Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.

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

376.32

C₂₁H₁₂O₇

76823-03-5

Solid

Light yellow to yellow

525

488

O=C1OC2(C3=C(OC4=C2C=CC(O)=C4)C=C(O)C=C3)C5=C1C=C(C(O)=O)C=C5

Room temperature in continental US; may vary elsewhere.

4°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]. Jungmi Lee, et al. Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET). Sens Actuators B Chem. 2018 Mar;256:1093-1099.