ReadiLink™ Rapid Cy5 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction

Name: ReadiLink™ Rapid Cy5 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
Brand: AAT Bioquest
SKU: 1292
Price: 174 USD
Availability: InStock

Cy5 is one of the most popular fluorescent labeling dyes for preparing orange-red fluorescent bioconjugates. However, most of the commercial Cy5 labeling kits require intensive hands-on time. This Cy5 ReadiLink™ labeling kit is one of the most robust protein labeling kits for preparing Cy5-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 Cy5 dye provided in the kit is optimized for labeling ~50 µg antibody. This Cy5 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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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
ReadiLink™ Rapid Cy3 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	555	569	150000¹	0.15¹	0.07	0.073
ReadiLink™ Rapid Cy3 Antibody Labeling Kit Production Scale	555	569	150000¹	0.15¹	0.07	0.073
ReadiLink™ Rapid Cy7 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	756	779	250000	0.3	0.05	0.036
ReadiLink™ Rapid Cy7 Antibody Labeling Kit Production Scale	756	779	250000	0.3	0.05	0.036
ReadiLink™ Rapid FITC Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	491	516	73000	0.92	-	0.35
ReadiLink™ Rapid FITC Antibody Labeling Kit Production Scale	491	516	73000	0.92	-	0.35
ReadiLink™ Rapid XFD488 Antibody Labeling Kit Production Scale	499	520	71000	0.92¹	0.30	0.11
ReadiLink™ Rapid XFD488 Antibody Labeling Kit XFD488 Same Structure to Alexa Fluor™ 488	499	520	71000	0.92¹	0.30	0.11
ReadiLink™ Rapid XFD555 Antibody Labeling Kit Production Scale	553	568	150000	0.1¹	0.08	0.08
ReadiLink™ Rapid XFD555 Antibody Labeling Kit XFD555 Same Structure to Alexa Fluor™ 555	553	568	150000	0.1¹	0.08	0.08
ReadiLink™ Rapid XFD594 Antibody Labeling Kit Production Scale	590	618	90000	0.66¹	0.43	0.56
ReadiLink™ Rapid XFD594 Antibody Labeling Kit XFD594 Same Structure to Alexa Fluor™ 594	590	618	90000	0.66¹	0.43	0.56
ReadiLink™ Rapid XFD647 Antibody Labeling Kit Production Scale	650	671	239000	0.33¹	0.00	0.03
ReadiLink™ Rapid XFD647 Antibody Labeling Kit XFD647 Same Structure to Alexa Fluor™ 647	650	671	239000	0.33¹	0.00	0.03
ReadiLink™ Rapid XFD750 Antibody Labeling Kit Production Scale	752	776	240000	0.12¹	0.00	0.04
ReadiLink™ Rapid XFD750 Antibody Labeling Kit XFD750 Same Structure to Alexa Fluor™ 750	752	776	240000	0.12¹	0.00	0.04
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Citations

View all 12 citations: Citation Explorer

Thermo-sensitive hydrogel PLGA-PEG-PLGA as a vaccine delivery system for intramuscular immunization
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

Cube-shaped theranostic paclitaxel prodrug nanocrystals with surface functionalization of SPC and MPEG-DSPE for imaging and chemotherapy
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)

Light/magnetic hyperthermia triggered drug released from multi-functional thermo-sensitive magnetoliposomes for precise cancer synergetic theranostics
Authors: Guo, Yuxin and Zhang, Yang and Ma, Jinyuan and Li, Qi and Li, Yang and Zhou, Xinyi and Zhao, Dan and Song, Hua and Chen, Qing and Zhu, Xuan
Journal: Journal of Controlled Release (2017)

Carboxymethyl Dextran-Stabilized Polyethylenimine-Poly (epsilon-caprolactone) Nanoparticles-Mediated Modulation of MicroRNA-34a Expression via Small-Molecule Modulator for Hepatocellular Carcinoma Therapy
Authors: Deng, Xiongwei and Yin, Zhaoxia and Zhou, Zhixiang and Wang, Yihui and Zhang, Fang and Hu, Qin and Yang, Yishu and Lu, Jianqing and Wu, Yan and Sheng, Wang and others, undefined
Journal: ACS applied materials & interfaces (2016): 17068--17079

Affinity-Controlled Protein Encapsulation into Sub-30 nm Telodendrimer Nanocarriers by Multivalent and Synergistic Interactions
Authors: Wang, Xu and Shi, Changying and Zhang, Li and Bodman, Alexa and Guo, D and an , undefined and Wang, Lili and Hall, Walter A and Wilkens, Stephan and Luo, Juntao
Journal: Biomaterials (2016)

References

View all 21 references: Citation Explorer

Excitation of Cy5 in self-assembled lipid bilayers using optical microresonators
Authors: Freeman LM, Li S, Dayani Y, Choi HS, Malmstadt N, Armani AM.
Journal: Appl Phys Lett (2011): 143703

Theranostic cRGD-BioShuttle Constructs Containing Temozolomide- and Cy7 For NIR-Imaging and Therapy
Authors: Wiessler M, Hennrich U, Pipkorn R, Waldeck W, Cao L, Peter J, Ehemann V, Semmler W, Lammers T, Braun K.
Journal: Theranostics (2011): 381

Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging
Authors: Berezin MY, Guo K, Akers W, Livingston J, Solomon M, Lee H, Liang K, Agee A, Achilefu S.
Journal: Biochemistry (2011): 2691

In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium
Authors: Adler ED, Bystrup A, Briley-Saebo KC, Mani V, Young W, Giovanonne S, Altman P, Kattman SJ, Frank JA, Weinmann HJ, Keller GM, Fayad ZA.
Journal: JACC Cardiovasc Imaging (2009): 1114

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine-based or three rhodamine-based dyes
Authors: Volke D, Hoffmann R.
Journal: Electrophoresis (2008): 4516

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Spectral properties

Correction Factor (260 nm)	0.02
Correction Factor (280 nm)	0.03
Correction Factor (482 nm)	0.009
Correction Factor (565 nm)	0.09
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	651
Emission (nm)	670
Quantum yield	0.27¹, 0.4²