Cy5 maleimide  (Synonyms: Cyanine5 maleimide)

Cy5 maleimide (Cyanine5 maleimide) is a single-reactive dye containing a maleimide group and a CY5 fluorescent group, which can selectively couple with a thiol group. Cy5 maleimide can be used to label antibodies, peptides, or proteins (Ex/Em = 646/664 nm).

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 6.5 ± 0.5.
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 Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well.
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 4.3 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 ×641.24 mg/mmol/0.01 mg/μL = 4.3 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.
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 1 M 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.

641.24

C₃₈H₄₅ClN₄O₃

1437796-65-0

Solid

Brown to dark brown

664

646

O=C(NCCN(C1=O)C(C=C1)=O)CCCCCN(/C2=C/C=C/C=C/C(C3(C)C)=[N+](C4=CC=CC=C34)C)C5=CC=CC=C5C2(C)C.[Cl-]

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.

• [1]. Maeda K, et al. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms. Biochem J. 2004 Mar 1;378(Pt 2):497-507.  [Content Brief]

  [2]. Ptaszek M. Rational design of fluorophores for 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. doi:10.1016/j.dyepig.2017.06.029