ReadiLink™ xtra Rapid Cy5 Antibody Labeling Kit BSA-Compatible

Immunofluorescence staining of tubulin in HeLa cells. HeLa cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked. Cells were then incubated with mouse anti-tubulin monoclonal antibody and stained with a goat anti-mouse IgG labeled using the ReadiLink™ xtra Rapid Cy5 Antibody Labeling Kit (Cat No. 1972). The cell nuclei were counterstained using DAPI (Cat No. 17507).

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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™ xtra Rapid iFluor® 647 Antibody Labeling Kit *BSA-Compatible*

Overview SDS Protocol

Upgrade: ReadiLink™ xtra Rapid iFluor® 647 Antibody Labeling Kit *BSA-Compatible*

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

ReadiLink™ xtra rapid antibody labeling kits require essentially only 2 simple mixing steps without a column purification needed. Specially formulated Cy5 used in this ReadiLink™ kit is quite stable and shows good reactivity and selectivity with antibodies. The kit has all the essential components for labeling ~2x50 ug antibody. Each of the two vials of specially formulated Cy5 dye provided in the kit is optimized for labeling ~50 µg antibody. ReadiLink™ xtra Cy5 rapid antibody labeling kit provides a convenient and robust method to label monoclonal and polyclonal antibodies with the red fluorescent Cy5 fluorophore. Cy5 is one of the most used fluorophores for labeling antibodies.

Figure 1. Overview of the ReadiLink™ xtra 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) with 0.1 to 0.5 % will 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™ xtra Rapid FITC Antibody Labeling Kit BSA-Compatible	491	516	73000	0.92
ReadiLink™ xtra Rapid Cy3 Antibody Labeling Kit BSA-Compatible	555	569	150000¹	0.15¹
ReadiLink™ xtra Rapid Cy7 Antibody Labeling Kit BSA-Compatible	756	779	250000	0.3
ReadiLink™ xtra Rapid XFD594 Antibody Labeling Kit BSA-Compatible, XFD594 Same Structure to Alexa Fluor™ 594	590	618	92000	0.66¹
ReadiLink™ xtra Rapid XFD488 Antibody Labeling Kit BSA-Compatible, XFD488 Same Structure to Alexa Fluor™ 488	499	520	73000	0.92¹
ReadiLink™ xtra Rapid XFD555 Antibody Labeling Kit BSA-Compatible, XFD555 Same Structure to Alexa Fluor™ 555	553	568	155000	0.1¹
ReadiLink™ xtra Rapid XFD647 Antibody Labeling Kit BSA-Compatible, XFD647 Same Structure to Alexa Fluor™ 647	650	671	270000	0.33¹
ReadiLink™ xtra Rapid XFD750 Antibody Labeling Kit BSA-Compatible, XFD750 Same Structure to Alexa Fluor™ 750	752	776	290000	0.12¹

Images

Figure 1. Immunofluorescence staining of tubulin in HeLa cells. HeLa cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked. Cells were then incubated with mouse anti-tubulin monoclonal antibody and stained with a goat anti-mouse IgG labeled using the ReadiLink™ xtra Rapid Cy5 Antibody Labeling Kit (Cat No. 1972). The cell nuclei were counterstained using DAPI (Cat No. 17507).

References

View all 50 references: Citation Explorer

Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes.
Authors: Nödling, Alexander R and Mills, Emily M and Li, Xuefei and Cardella, Davide and Sayers, Edward J and Wu, Shih-Hsiung and Jones, Arwyn T and Luk, Louis Y P and Tsai, Yu-Hsuan
Journal: Chemical communications (Cambridge, England) (2020): 4672-4675

Hybrid Tracers Based on Cyanine Backbones Targeting Prostate-Specific Membrane Antigen: Tuning Pharmacokinetic Properties and Exploring Dye-Protein Interaction.
Authors: Hensbergen, Albertus W and Buckle, Tessa and van Willigen, Danny M and Schottelius, Margret and Welling, Mick M and van der Wijk, Felicia A and Maurer, Tobias and van der Poel, Henk G and van der Pluijm, Gabri and van Weerden, Wytske M and Wester, Hans-Jürgen and van Leeuwen, Fijs W B
Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (2020): 234-241

Direct Cyanine-dCTP Labeling of DNA for Microarrays.
Authors: Rando, Oliver
Journal: Cold Spring Harbor protocols (2019)

Direct Cyanine-dUTP Labeling of RNA for Microarrays.
Authors: Rando, Oliver
Journal: Cold Spring Harbor protocols (2019)

Electrochemiluminescent aptasensor based on resonance energy transfer system between CdTe quantum dots and cyanine dyes for the sensitive detection of Ochratoxin A.
Authors: Gao, Jingwen and Chen, Ziyi and Mao, Lebao and Zhang, Wei and Wen, Wei and Zhang, Xiuhua and Wang, Shengfu
Journal: Talanta (2019): 178-183

Computational and experimental data on electrostatic density and stacking tendency of asymmetric cyanine 5 dyes.
Authors: Spa, S J and Hensbergen, A W and van der Wal, S and Kuil, J and van Leeuwen, F W B
Journal: Data in brief (2019): 50-55

Harnessing Cyanine Reactivity for Optical Imaging and Drug Delivery.
Authors: Gorka, Alexander P and Nani, Roger R and Schnermann, Martin J
Journal: Accounts of chemical research (2018): 3226-3235

Tracers for Fluorescence-Guided Surgery: How Elongation of the Polymethine Chain in Cyanine Dyes Alters the Pharmacokinetics of a Dual-Modality c[RGDyK] Tracer.
Authors: Buckle, Tessa and van Willigen, Danny M and Spa, Silvia J and Hensbergen, Albertus W and van der Wal, Steffen and de Korne, Clarize M and Welling, Mick M and van der Poel, Henk G and Hardwick, James C H and van Leeuwen, Fijs W B
Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (2018): 986-992

Self-Healing Organic Fluorophore of Cyanine-Conjugated Amphiphilic Polypeptide for Near-Infrared Photostable Bioimaging.
Authors: Li, Tuanwei and Liu, Le and Jing, Titao and Ruan, Zheng and Yuan, Pan and Yan, Lifeng
Journal: ACS applied materials & interfaces (2018): 14517-14530

A ruthenium(ii) complex-cyanine energy transfer scaffold based luminescence probe for ratiometric detection and imaging of mitochondrial peroxynitrite.
Authors: Zhang, Wenzhu and Liu, Yi and Gao, Quankun and Liu, Chaolong and Song, Bo and Zhang, Run and Yuan, Jingli
Journal: Chemical communications (Cambridge, England) (2018): 13698-13701