ReadiLink™ Rapid Cy3 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
Cy3 is one of the most popular fluorescent labeling dyes for preparing orange-red fluorescent bioconjugates. However, most of the commercial Cy3 labeling kits require intensive hands-on time. This Cy3 ReadiLink™ labeling kit is one of the most robust protein labeling kits for preparing Cy3-labeled antibody conjugates or other protein conjugates. It essentially only requires 2 simple mixing steps without a column purification required. The kit provides all the essential components for labeling ~2x50 ug antibody. Each of the two vials of Cy3 dye provided in the kit is optimized for labeling ~50 µg antibody. This Cy3 protein labeling kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa).
Figure 1. Overview of the ReadiLink™ Rapid Antibody Labeling protocol. In just two simple steps, and with no purification necessary, covalently label microgram amounts of antibodies in under an hour.
Example protocol
AT A GLANCE
Important
Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following protocol is for recommendation.PREPARATION OF WORKING SOLUTION
Protein working solution (Solution A)
For labeling 50 µg of protein (assuming the target protein concentration is 1 mg/mL), mix 5 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 50 µL of the target protein solution.Note If you have a different protein concentration, adjust the protein volume accordingly to make ~50 µg of protein available for your labeling reaction.
Note For labeling 100 µg of protein (assuming the target protein concentration is 1 mg/mL), mix 10 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 100 µL of the target protein solution.
Note The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2 - 7.4; if the protein is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2 - 7.4, or use Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa (cat# UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
Note For optimal labeling efficiency, a final protein concentration range of 1 - 2 mg/mL is recommended, with a significantly reduced conjugation efficiency at less than 1 mg/mL.
SAMPLE EXPERIMENTAL PROTOCOL
Run conjugation reaction
- Add the protein working solution (Solution A) to ONE vial of labeling dye (Component A), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
Note If labeling 100 µg of protein, use both vials (Component A) of labeling dye by dividing the 100 µg of protein into 2 x 50 µg of protein and reacting each 50 µg of protein with one vial of labeling dye. Then combine both vials for the next step. - Keep the conjugation reaction mixture at room temperature for 30 - 60 minutes.
Note The conjugation reaction mixture can be rotated or shaken for longer time if desired.
Stop Conjugation reaction
- Add 5 µL (for 50 µg protein) or 10 µL (for 100 µg protein) which is 10% of the total reaction volume of TQ™-Dyed Quench Buffer (Component C) into the conjugation reaction mixture; mix well.
- Incubate at room temperature for 10 minutes. The labeled protein (antibody) is now ready to use.
Storage of Protein Conjugate
The protein conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). For longer storage, the protein conjugates could be lyophilized or divided into single-used aliquots and stored at ≤ –20°C.Spectrum
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Citations
View all 14 citations: Citation Explorer
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Authors: Kim, Ji Hong and Jeong, Hye-Seon and Hwang, Jaehyeon and Kweon, Dae-Hyuk and Choi, Chang-Hyung and Park, Jong Pil
Journal: ACS Applied Materials \& Interfaces (2023)
Authors: Kim, Ji Hong and Jeong, Hye-Seon and Hwang, Jaehyeon and Kweon, Dae-Hyuk and Choi, Chang-Hyung and Park, Jong Pil
Journal: ACS Applied Materials \& Interfaces (2023)
Antibody-antibiotic conjugate targeted therapy for orthopedic implant-associated intracellular S. aureus infections
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Thermo-sensitive hydrogel PLGA-PEG-PLGA as a vaccine delivery system for intramuscular immunization
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Authors: Wang, Xiaoyan and Zhang, Yu and Xue, Wei and Wang, Hong and Qiu, Xiaozhong and Liu, Zonghua
Journal: Journal of Biomaterials Applications (2017): 923--932
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Authors: Guo, Fuqiang and Shang, Jiajia and Zhao, Hai and Lai, Kangrong and Li, Yang and Fan, Zhongxiong and Hou, Zhenqing and Su, Guanghao
Journal: Colloids and Surfaces B: Biointerfaces (2017)
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Journal: Colloids and Surfaces B: Biointerfaces (2017)
Light/magnetic hyperthermia triggered drug released from multi-functional thermo-sensitive magnetoliposomes for precise cancer synergetic theranostics
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Journal: Journal of Controlled Release (2017)
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Journal: Journal of Controlled Release (2017)
References
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