Cy7.5 maleimide specifically targets free thiol groups (-SH, sulfhydryl groups) on proteins, peptides, and antibodies. The maleimide group reacts rapidly and selectively with thiols at pH 6.5-7.5, forming a stable thioether bond. This reaction enables site-specific labeling of reduced disulfide bonds or naturally occurring cysteine residues in biomolecules. The compound is not a pharmaceutical drug but a chemical labeling tool for fluorescence-based detection and imaging applications.

The finest stock solution preparation 1. Making protein A 2 mg/mL protein (antibody) concentration is added to achieve the labeling effect. A pH of 8.5±0.5 is the ideal value for the protein solution. Use one milligram of carbon dioxide if the pH is less than 8.0. The labeling efficiency will be significantly decreased if the protein content is less than 2 mg/mL. Ultimately, a protein concentration range of 2–10 mg/mL is advised to achieve the optimal labeling efficiency. The labeling efficiency of the protein will be impacted if it is not placed in a transparent buffer that contains primary amines (like Tris or glycine) and ammonium ions. 2. Prepare the dye Establish a 10 mM stock solution by mixing CY dye with anhydrous DMSO. Before being aliquoted, the CY storage solution should be properly mixed using a glass tube or vortex and kept in a dark place at -20°C or -80°C. 3. The quantity of dye solution used Depending on how much protein has been labeled, CY dye will be needed for the labeling procedure. CY dye and protein should be taken at the following dosages: If 500 μL of 2 mg/mL IgG (MW = 150.000) is the necessary labeled protein, then 3.95 μL of CY volume is needed. To dissolve a tube containing 1 milligram of CY dye, use 100 μL of DMSO. To be more specific, the calculation procedure goes like this (using CY3-NHS ester as an example). 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 Three) μL of CY3-NHS ester equals mmol of CY3-NHS ester, ×MW of CY3-NHS ester, / mg/μL of CY3-NHS ester, which translates to 6.7×10-5 mmol× 590.15 mg/mmol / 0.01 mg/μL, or 3.95 μL (by volume). Method of use 1. Reaction labeling 1) Slowly fill the fresh carrier with the calculated volume of 10 mg/mL CY dye. The centrifuged sample should be briefly collected at the bottom of the reaction tube. Add to the 0.5 mL protein sample solution, shake gently to mix. Never copy 2) Place the reaction tube in a dark area, give it a good shake, and walk for 60 minutes under the original settings (10–15 minutes each week). Gently flip the reaction tube over multiple times to reach 2. Protein desalting and blocking SepHadex G-25 column blocked dye conjugates are used as an example in the methodology that follows. Read the manufacturer's instructions and prepare the SepHadex G-25 column. 2) Fill the top of the SepHadex G-25 column with the reaction mixture. Upon reaching the lower limit of the upper resin's surface, incorporate PBS (pH 7.2-7.4). To complete columnar shrinking, apply extra PBS (pH 7.2–7.4) right away to the required sample. To create the desired dye-protein conjugate, combine the components.

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In vitro, Cy7.5 maleimide is used to label thiol-containing proteins for a variety of fluorescence detection methods. The near-infrared emission at 820 nm allows for deep tissue penetration and reduced background autofluorescence in biological samples. For protein labeling, the maleimide group reacts specifically with cysteine thiols at pH 6.5-7.5. Optimal labeling conditions require the protein to be in a buffer free of primary amines (such as Tris or glycine) and at a concentration of 2-10 mg/mL. The dye is dissolved in anhydrous DMSO to a 10 mM stock solution. A 10-fold molar excess of dye is typically added to achieve a labeling degree of 2-5 dyes per protein molecule.

In vivo, Cy7.5 maleimide is not administered directly but is used to pre-label biomolecules for near-infrared fluorescence imaging in live animals. The dye-conjugated biomolecules retain their biological activity, and the NIRF properties allow non-invasive imaging of tissue distribution, tumor targeting, and biodistribution in living mice (Ex/Em: 770/820 nm). The 820 nm emission minimizes light scattering and absorption by hemoglobin and water, enabling imaging depths of up to 1-2 cm.

For non-cell-based conjugation assays, a standard protocol is used to conjugate Cy7.5 maleimide to a thiol-containing protein. Protein (2-10 mg/mL) is prepared in PBS (pH 7.2) or 100 mM phosphate buffer (pH 7.0). If disulfide bonds need to be reduced, treat with 5-10 mM TCEP for 30 minutes at room temperature, followed by buffer exchange. The dye is dissolved in anhydrous DMSO to 10 mM. A 10-20 fold molar excess of dye is added to the protein solution and reacted for 2 hours at room temperature or overnight at 4degC, protected from light. Unconjugated dye is removed by size exclusion chromatography (Sephadex G-25) or dialysis. Labeling degree is determined by absorbance at 280 nm and 770 nm.

For in vitro cell-based assays, cells (e.g., HeLa, MDA-MB-231) are seeded in glass-bottom culture dishes or 96-well plates. A protein of interest (e.g., antibody, transferrin) is labeled with Cy7.5 maleimide using the protocol described. Cells are incubated with the labeled protein (typically 1-50 ug/mL) in culture medium for 30-60 minutes at 37degC. For live-cell imaging, cells are washed with PBS to remove unbound label and maintained in phenol-red-free medium. Images are acquired using a confocal microscope equipped with a 785 nm laser and 820 nm emission filter. For fixed-cell imaging, cells are washed, fixed with 4% paraformaldehyde, and mounted.

For in vivo animal studies, a mouse xenograft tumor model is used. Nude mice (6-8 weeks old) bearing subcutaneous tumor xenografts (100-200 mm3) are administered a Cy7.5-labeled tumor-targeting antibody (e.g., anti-HER2 or anti-EGFR) via tail vein injection at 100-200 ug of labeled antibody in 100-200 uL PBS. Mice are anesthetized with 2% isoflurane, and NIR fluorescence images are acquired at 0, 6, 12, 24, 48, 72 hours post-injection using an in vivo imaging system with 770 nm excitation and 820 nm emission filters. Fluorescence intensity in tumor region is quantified. At final time point, organs are harvested for ex vivo imaging.

Cy7.5 maleimide has a molecular weight of 807.46 g/mol and excitation/emission maxima of 770 nm/820 nm, making it suitable for deep tissue near-infrared imaging. The compound is soluble in DMSO and can be dissolved to 10 mM for stock solutions. For protein labeling, the working solution should be prepared immediately before use as the maleimide group is prone to hydrolysis. The dye should be stored at -20degC, protected from light and moisture. In solution, it is stable at -80degC for 6 months or -20degC for 1 month when sealed and protected from moisture and light.

The maleimide group is chemically reactive and can cause skin and eye irritation upon direct contact. Standard chemical safety practices should be followed: wear gloves, a lab coat, and safety goggles when handling this compound. The compound is not intended for human therapeutic or diagnostic use. No specific acute toxicity data is available, but the compound should be handled as a potential irritant. At the concentrations used for labeling (uM to low mM), no significant toxicity is observed in cell culture. Hydrolysis of the maleimide group generates N-hydroxysuccinimide, which is of low toxicity.

[1]. Biodegradable Zwitterionic Nanogels with Long Circulation for Antitumor Drug Delivery. ACS Appl Mater Interfaces. 2018 Jul 18;10(28):23509-23521.

[2]. Ptaszek M. Rational design of fluorophores for in vivo applications. Prog Mol Biol Transl Sci. 2013;113:59-108.

[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.

Cy7.5 maleimide is a member of the cyanine dye family, characterized by two nitrogen atoms connected by an odd number of methyl units, giving these dyes long absorption/emission wavelengths, high extinction coefficients, and good water solubility. The maleimide functional group provides selective reactivity with thiol groups, which is particularly useful for labeling antibodies and proteins at specific sites (e.g., reduced hinge region cysteine residues). This dye is widely used in fluorescence microscopy, flow cytometry, Western blotting, and in vivo imaging for research purposes. The product is for research use only.

C51H55CLN4O3

807.46

806.396

2270866-73-2

162477420

Green to dark green solid powder

1

5

12

59

1760

0

C1=CC=CC2=C3C(N(C)/C(=C\C=C4/CCCC(C=CC5=[N+](CCCCCC(NCCN6C(=O)C=CC6=O)=O)C6=CC=C7C=CC=CC7=C6C5(C)C)=C/4)/C3(C)C)=CC=C12.[Cl-] |t:17|

XCNYYRVWJSOPSW-UHFFFAOYSA-N

InChI=1S/C51H54N4O3.ClH/c1-50(2)43(53(5)41-25-23-37-16-8-10-18-39(37)48(41)50)27-21-35-14-13-15-36(34-35)22-28-44-51(3,4)49-40-19-11-9-17-38(40)24-26-42(49)54(44)32-12-6-7-20-45(56)52-31-33-55-46(57)29-30-47(55)58;/h8-11,16-19,21-30,34H,6-7,12-15,20,31-33H2,1-5H3;1H

6-[(2Z)-1,1-dimethyl-2-[(2Z)-2-[3-[(E)-2-(1,1,3-trimethylbenzo[e]indol-3-ium-2-yl)ethenyl]cyclohex-2-en-1-ylidene]ethylidene]benzo[e]indol-3-yl]-N-[2-(2,5-dioxopyrrol-1-yl)ethyl]hexanamide;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)