AF488 NHS ester TEA 

AF488 NHS ester TEA is an amine specific fluorescence probe (λ_em=525 nm=525 nm). AF488 NHS ester reacts with sulfhydryl groups and amines in aqueous and biological samples then change their chemical structure and fluorescence properties after derivatization. AF488 exhibits the λ_em and λ_ex wavelength of 520 nm and 470 nm, respectively^[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 AF488 NHS ester TEA to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of AF488 NHS ester TEA required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of AF488 NHS ester TEA 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 AF488 NHS ester TEA, the required AF488 NHS ester TEA volume is 4.91 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 (AF488 NHS ester TEA) = mmol (IgG) × 10 = 6.7×10^-6 mmol×10 = 6.7 × 10^-5 mmol
3) μL (AF488 NHS ester TEA) = mmol (AF488 NHS ester TEA) ×MW (AF488 NHS ester TEA)/mg/μL (AF488 NHS ester TEA) = 6.7 ×10^-5 mmol ×732.73 mg/mmol/0.01 mg/μL = 4.91 μL (AF488 NHS ester TEA)
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL AF488 NHS ester TEA 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.

732.73

C₃₁H₃₂N₄O₁₃S₂

Solid

Orange to red

O=C(C1=C(C2=C(C=CC(N)=C3S(=O)(O)=O)C3=[O+]C4=C2C=CC(N)=C4S(=O)([O-])=O)C=CC(C(ON5C(CCC5=O)=O)=O)=C1)O.CCN(CC)CC

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.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

• [1]. Korlach J, et al. Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. Nucleosides Nucleotides Nucleic Acids. 2008 Sep;27(9):1072-83.  [Content Brief]

  [2]. Chakraborty S, Hu S Y, Wu S H, et al. The interaction affinity between vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4) analyzed by quantitative FRET[J]. PLoS One, 2015, 10(3): e0121399.