Alexa Fluor 680 NHS ester diTEA  (Synonyms: Alexa Fluor 680 succinimidyl ester diTEA)

Alexa Fluor 680 NHS ester (Alexa Fluor 680 succinimidyl ester) diTEA is a near-infrared (NIR) fluorescent dye. NHS esters can be used to label to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules (Ex/Em = 679/702 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 Alexa Fluor 680 NHS ester diTEA to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of Alexa Fluor 680 NHS ester diTEA required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of Alexa Fluor 680 NHS ester diTEA 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 Alexa Fluor 680 NHS ester diTEA, the required Alexa Fluor 680 NHS ester diTEA volume is 7.76 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 (Alexa Fluor 680 NHS ester diTEA) = mmol (IgG) × 10 = 6.7×10^-6 mmol×10 = 6.7 × 10^-5 mmol
3) μL (Alexa Fluor 680 NHS ester diTEA) = mmol (Alexa Fluor 680 NHS ester diTEA) ×MW (Alexa Fluor 680 NHS ester diTEA)/mg/μL (Alexa Fluor 680 NHS ester diTEA) = 6.7 ×10^-5 mmol × 1158.29 mg/mmol / 0.01 mg/μL = 7.76 μL (Alexa Fluor 680 NHS ester diTEA)
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL Alexa Fluor 680 NHS ester diTEA 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.

1158.29

Alexa Fluor 680 succinimidyl ester diTEA

C₅₁H₇₇BrN₆O₁₃S₃

407628-16-4

O=C(ON1C(CCC1=O)=O)CCCCC[C@]2(C3=CC(S(=O)(O)=O)=CC=C3N(/C2=C\C=C\C=C\C4=NC5=[N+](C=C(C=C5C4(C)C)Br)CCCS(=O)([O-])=O)CCCS(=O)(O)=O)C.CCN(CC)CC.CCN(CC)CC

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Cheng KT, et al. Alexa Fluor 680-labeled transferrin-cationic (NBD-labeled DOPE-DOTAP) liposome-encapsulated gadopentetate dimeglumine complex. 2007 Oct 18 [updated 2007 Dec 17]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.  [Content Brief]