5-FAM SE 

5-FAM SE is a single isomer, is a fluorescent labeling reagent used for labeling peptides, proteins and nucleotides. 5-FAM SE can react with amines and can yield stable amine conjugates^[1][2].

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 SE to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of 5-FAM SE 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 SE, the required 5-FAM SE volume is 3.17 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 SE) = mmol (IgG) × 10 = 6.7×10^-6 mmol×10 = 6.7 × 10^-5 mmol
3) μL (5-FAM SE) = mmol (5-FAM SE) ×MW (5-FAM SE)/mg/μL (5-FAM SE) = 6.7 ×10^-5 mmol ×473.39 mg/mmol / 0.01 mg/μL = 3.17 μL (5-FAM SE)
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL 5-FAM SE 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.

473.39

C₂₅H₁₅NO₉

92557-80-7

Solid

Yellow to orange

515

488

O=C(C1=CC2=C(C3(C4=C(OC5=C3C=CC(O)=C5)C=C(O)C=C4)OC2=O)C=C1)ON6C(CCC6=O)=O

Room temperature in continental US; may vary elsewhere.

-20°C, protect from light, stored under nitrogen

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

* 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, stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Sheng-da Qi, et al. Quantification of luminally released serotonin in rat proximal colon by capillary electrophoresis with laser-induced fluorescence detection. Anal Bioanal Chem. 2009 Apr;393(8):2059-66.  [Content Brief]

  [2]. Xiao-Ling Zhang, et al. Sensitive determination of pheomelanin after 5-carboxyfluorescein succinimidyl ester precapillary derivatization and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Jan 1;861(1):136-9.  [Content Brief]