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].

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Protein Preparation
To obtain the best labeling effect, please prepare the protein (antibody) concentration to 2 mg/mL.
1.1 The pH value of the protein solution should be 8.5 ± 0.5. If the pH is lower than 8.0, adjust it with 1 M sodium bicarbonate.
1.2 If the protein concentration is lower than 2 mg/mL, the labeling efficiency will be greatly reduced. To obtain the best labeling efficiency, it is recommended that the final protein concentration range be 2-10 mg/mL.
1.3 The protein must be in a buffer free of primary amines (such as Tris or glycine) and ammonium ions, otherwise the labeling efficiency will be affected.
2. Dye Preparation
Dilute the dye with anhydrous DMSO to prepare a clear stock solution of 10 mg/mL.
3. Calculation of Dye Amount
The amount of dye required for the reaction depends on the amount of protein to be labeled. The optimal molar ratio of dye to protein is approximately 10:1.
Example: Assuming the required labeled protein is 500 μL 2 mg/mL IgG (MW=150,000), and 1 mg of dye is dissolved in 100 μL DMSO, the required dye volume is 4.9 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 (dye) = mmol (IgG) × 10 = 6.7 × 10^-6 mmol × 10 = 6.7 × 10^-5 mmol
3) μL (dye) = mmol (dye) × MW (dye)/mg/μL (dye) = 6.7 ×10^-5 mmol ×732.73 mg/mmol/0.01 mg/μL = 4.9 μL (dye)
Note: We recommend using a dye:protein molar ratio of 10:1. If the concentration is too low or too high, the ratio can be adjusted to 5:1, 15:1, or 20:1.
4. Running the Coupling Reaction
1) Slowly add the calculated volume of freshly prepared 10 mg/mL dye to 0.5 mL of protein sample solution, gently shake to mix, and then briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid vigorous mixing to prevent denaturation and inactivation of the protein sample.
2) Place the reaction tube in a dark place and incubate gently with shaking for 60 minutes at room temperature. Every 10-15 minutes, gently invert the reaction tube several times to thoroughly mix the reactants and improve labeling efficiency.
5. Purification of Conjugates
The following protocol is an example of purifying dye-protein conjugates using a Labelling Kits Centrifugation-Based Rapid Desalting Column (5KD) (HY-D3014).
5.1 Prepare the desalting column according to the instructions.
5.2 Load the reaction mixture onto the top of the desalting column.
5.3 Once the sample has run below the resin surface, immediately add PBS (pH 7.2-7.4).
5.4 Add more PBS (pH 7.2-7.4) to the target sample to complete column purification. Collect the fraction containing 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.

-20°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.