Cy3B NHS ester

Cy3B NHS ester is a fluorescent dye compound that is commonly used in biomarking and fluorescent labeling experiments, especially for labeling biomolecules containing amino functional groups (amine groups), such as proteins, antibodies or peptides. In Vitro: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 (Example for Cy3B NHS ester)
Add anhydrous DMSO into the vial of Cy3B NHS ester to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of Cy3B NHS ester required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of Cy3B NHS ester 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 Cy3B NHS ester, the required Cy3B NHS ester volume is 4.4 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 (Cy3B NHS ester) = mmol (IgG) × 10 = 6.7×10^-6 mmol×10 = 6.7 × 10^-5 mmol
3) μL (Cy3B NHS ester) = mmol (Cy3B NHS ester) ×MW (Cy3B NHS ester)/mg/μL (Cy3B NHS ester) = 6.7 ×10^-5 mmol × 657.73 mg/mmol/0.01 mg/μL = 4.4 μL (Cy3B NHS ester)
Note: We recommend using a 10:1 molar ratio of dye/protein. If it is too low or too high, determine the optimal dye/protein ratio at 5:1, 15:1, and 20:1, respectively.
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL Cy3B NHS ester 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.