Cyanine 3.5 maleimide chloride 

Cyanine3.5 maleimide chloride is a CY dye. CY, short for Cyanine, is a compound consisting of two nitrogen atoms connected by an odd number of methyl units. Cyanine compounds have the characteristics of long wavelength, adjustable absorption and emission, high extinction coefficient, good water solubility and relatively simple synthesis^[1]. CY dyes are of en used for the labeling of proteins, antibodies and small molecular compounds. For the labeling of protein antibodies, the combination can be completed through a simple mixing reaction. Below, we introduce the labeling method of protein antibody labeling, which has certain reference significance^[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.
2.Dye Preparation (Example for CY3-NHS ester)
Add anhydrous DMSO into the vial of CY3-NHS ester to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of CY3-NHS ester required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of CY3-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 CY3-NHS ester, the required CY3-NHS ester volume is 5.05 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 (CY3-NHS ester) = mmol (IgG) × 10 = 6.7×10-6 mmol×10 = 6.7 × 10-5 mmol
3) μL (CY3-NHS ester) = mmol (CY3-NHS ester) ×MW (CY3-NHS ester) / mg/μL (CY3-NHS ester) = 6.7 ×10-5 mmol ×753.88 mg/mmol / 0.01 mg/μL = 5.05 μL (CY3-NHS ester)
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL CY3-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.

Optional
If the protein does not contain free cysteines, the protein must be treated with DTT or TCEP to generate thiol groups. DTT or TCEP converts disulfide bonds to two free thiol groups. If DTT is used, free DTT must be removed by dialysis or gel filtration prior to conjugation of the maleimide dye to the protein. The following is an example protocol for generating free thiol groups:
1. Prepare a fresh solution of 1M DTT (15.4 mg/100 µL) in distilled water.
2. Prepare an IgG solution in 20 mM DTT: add 20 µL of DTT stock solution per ml of IgG solution while mixing. Let stand at room temperature for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystine).
3. Pass the reduced IgG through a filter column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions from the column.
4. Determine protein concentration and pool the fractions containing the most IgG. This can be done spectrophotometrically or colorimetrically.
Perform conjugation as soon as possible after this step (see example protocol).
Note: For good results, the IgG solution should be >4 mg/mL. If the antibody is below 2 mg/mL, it should be concentrated. Additionally, a 10% increase is necessary to compensate for losses on the buffer exchange column.
Note: The reduction reaction can be performed in any buffer at pH 7-7.5, such as MES, phosphate, or TRIS buffer.
Note: Steps 3 and 4 can be replaced by dialysis.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

715.32

C₄₄H₄₇ClN₄O₃

3077081-59-2

O=C1C=CC(N1CCNC(CCCCCN2C3=C(C(C)(C)/C2=C\C=C\C4=[N+](C)C5=C(C4(C)C)C6=C(C=CC=C6)C=C5)C7=C(C=CC=C7)C=C3)=O)=O.[Cl-]

Room temperature in continental US; may vary elsewhere.

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

• [1]. Masarapu H, et al. Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs. Biomacromolecules. 2017 Dec 11;18(12):4141-4153.  [Content Brief]

  [2]. or in vivo applications. Prog Mol Biol Transl Sci. 2013;113:59-108.  [Content Brief]

  [3]. Shindy, H. A. (2017). Fundamentals in the chemistry of cyanine dyes: A review. Dyes and Pigments, 145, 505–513.