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

HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT’s Cy5<sup>®</sup> goat anti-mouse IgG conjugate (Red, Left) or Jackson’s Cy5<sup>®</sup> goat anti-mouse IgG conjugate (Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

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

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Direct upgrades

ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*

Overview SDS Protocol

Upgrade: ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*

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

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.

Components

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

Open in Advanced Spectrum Viewer

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

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield
ReadiLink™ Rapid FITC Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	491	516	73000	0.92
ReadiLink™ Rapid Cy3 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	555	569	150000¹	0.15¹
ReadiLink™ Rapid Cy7 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	756	779	250000	0.3
ReadiLink™ Rapid XFD488 Antibody Labeling Kit XFD488 Same Structure to Alexa Fluor™ 488	499	520	73000	0.92¹
ReadiLink™ Rapid XFD555 Antibody Labeling Kit XFD555 Same Structure to Alexa Fluor™ 555	553	568	155000	0.1¹
ReadiLink™ Rapid XFD594 Antibody Labeling Kit XFD594 Same Structure to Alexa Fluor™ 594	590	618	92000	0.66¹
ReadiLink™ Rapid XFD647 Antibody Labeling Kit XFD647 Same Structure to Alexa Fluor™ 647	650	671	270000	0.33¹
ReadiLink™ Rapid XFD750 Antibody Labeling Kit XFD750 Same Structure to Alexa Fluor™ 750	752	776	290000	0.12¹

Images

Figure 1. HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT’s Cy5^® goat anti-mouse IgG conjugate (Red, Left) or Jackson’s Cy5^® goat anti-mouse IgG conjugate (Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).

Citations

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Authors: Wang, Xiaoyan and Zhang, Yu and Xue, Wei and Wang, Hong and Qiu, Xiaozhong and Liu, Zonghua
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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)

Molecular Basis and Consequences of the Cytochrome c-tRNA Interaction
Authors: Liu, Cuiping and Stonestrom, Aaron J and Christian, Thomas and Yong, Jeongsik and Takase, Ryuichi and Hou, Ya-Ming and Yang, Xiaolu
Journal: Journal of Biological Chemistry (2016): 10426--10436

Click-electron microscopy for imaging metabolically tagged nonprotein biomolecules
Authors: Ngo, John T and Adams, Stephen R and Deerinck, Thomas J and Boassa, Daniela and Rodriguez-Rivera, Frances and Palida, Sakina F and Bertozzi, Carolyn R and Ellisman, Mark H and Tsien, Roger Y
Journal: Nat Chem Biol (2016): 459--465

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Authors: Zhao, Wen and Zhang, Yifan and Jiang, Xueyun and Cui, Chunying
Journal: Drug Design, Development and Therapy (2016): 3851

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)

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

Multiplexed single-cell in situ RNA analysis by reiterative hybridization
Authors: Xiao, Lu and Guo, Jia
Journal: Analytical Methods (2015): 7290--7295

Determination of the active transport of fucoidan derived from okinawa mozuku across the human intestinal caco-2 cells as assessed by size-exclusion chromatography
Authors: Nagamine, Takeaki and Hayakawa, Kou and Nakazato, Kyoumi and Iha, Masahiko
Journal: Journal of Chromatography B (2015): 187--193

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