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ReadiLink™ Rapid Cy3 Antibody Labeling Kit *Production Scale*

ReadiLink™ Rapid Antibody Labeling Kits, designed for production scale, provide a convenient and efficient method for labeling large volumes of antibodies with our superior iFluor® dyes, XFD dyes (equivalent to Alexa Fluor®), and various other labels. These kits utilize reactive fluorophores modified with succinimidyl ester (SE) functional groups, which selectively bind to primary amines on proteins, resulting in remarkably bright and photostable conjugates. Every kit contains all the necessary components for three distinct labeling reactions and features a user-friendly, pre-packed spin column for efficient dye removal, maximizing conjugate yield. Each vial of Cy3 dye provided in the kit is precisely formulated to label 1 mg of purified protein or antibody. Before labeling, it is important to remove stabilizing proteins like BSA from the sample and refrain from using amine-rich buffers like Tris, which might disrupt the labeling process. The Cy3 dye is a bright, orange-fluorescent dye with excitation and emission maxima of ~555 nm and ~569 nm, respectively. Cy3 conjugates are ideal for imaging, flow cytometry, and genomic applications. With ReadiLink™ Rapid Antibody Labeling kits, researchers can directly label primary antibodies, eliminating the need for secondary antibodies and enhancing panel-building flexibility.

Example protocol

AT A GLANCE

Key Parameters for Optimal Results
  1. 1.0 mg Antibody (MW ~150 kDa)

  2. Antibody concentration: 2.0 mg/mL

  3. Antibody volume: 500 µL

SAMPLE EXPERIMENTAL PROTOCOL

Important

Before opening the vials, warm all components and briefly centrifuge. Immediately prepare necessary solutions before starting conjugation. This protocol is a recommendation.

Antibody Labeling Reaction
  1. Warm up a vial of reactive dye (Component A) to room temperature.

    Note: Each vial of reactive dye contains an optimized amount of dye to label 1 mg of IgG (MW ~150 kDa) at 2 mg/mL in PBS, the kit can also be used to label other proteins (>10 kDa).

  2. Add 10 µL of DMSO (Component D) to the vial of reactive dye (Component A), mix well.

  3. Prepare a 500 µL antibody solution in PBS with a concentration of 2 mg/mL.

    Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2 - 7.4. If the protein is dissolved in buffers containing primary amines, like Tris and/or glycine, it must be dialyzed against 1X PBS, pH 7.2 - 7.4, or use Amicon Ultra0.5, Ultracel-10 Membrane, 10 kDa (Cat No. 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.

  4. Add 25 µL of Reaction Buffer (Component B) to the antibody solution.

  5. Transfer the reconstituted dye solution into the vial of antibody solution, and pipette several times to mix well.

  6. Rotate the reaction mixture for 1 hour at room temperature.

Purification with Desalting Column
  1. Twist off the bottom closure of the desalting column (Component D), and loosen the cap. Place the column in a collection tube.

  2. Centrifuge the column at 1,000 g for 2 minutes to remove the storage solution.

  3. Remove the cap and slowly add 1 mL of PBS to the column. Centrifuge at 1,000 g for 2 minutes and remove the buffer. Repeat this step 3 additional times, discarding the buffer from the collection tube each time.

  4. Place the column in a new collection tube, and gently apply the sample into the center of the compact resin bed.

  5. Centrifuge the column at 1,000 g for 2 minutes to collect the sample.

Determine the Antibody Concentration & Degree of Labeling (Optional)

The following formula can be used to calculate the antibody concentration:

(A280 - CF280 x Adye) / 1.4

The following formula can be used to calculate the degree of labeling:

DOL = (Adye / Ecdye) / (A280 - CF280 x Adye) / 210,000)

Where: 

  • 210,000 is the molar extinction coefficient (Ec) in cm-1M-1 of IgG at 280 nm.
  • CF280 is the correction factor for the effect of the fluorophore on absorbance at 280 nm.
  • Adye is the absorbance at maximum (λmax) for the respective dye.

Table 1. Properties of Labeling Dyes found in the ReadiLink™ Rapid Antibody Labeling Kits.

Cat#

Dye

Mol. Wt.

Ec (cm-1M-1)

CF280

Target DOL

5700

iFluor® 350

749.85

20,000

0.23

5-10

5702

iFluor® 488

945.07

75,000

0.21

4-8

5705

iFluor® 555

914.06

90,000

0.16

4-7

5710

iFluor® 594

1160.42

18,000

0.04

3-6

5713

iFluor® 647

1274.66

250,000

0.03

3-7

5718

iFluor® 750

1416.83

250,000

0.039

2-6

5720

FITC

620.52

75,000

0.183

3-6

5722

Cy3

829.03

150,000

0.073

1-3

5725

Cy5

855.07

250,000

0.03

2-4

5727

Cy7

881.11

250,000

0.036

2-4

5730

XFD488

643.4

71,000

0.11

4-8

5733

XFD555

1250

150,000

0.08

4-7

5736

XFD594

819.85

90,000

0.56

3-6

5740

XFD647

1259.66

240,000

0.03

3-7

5745

XFD750

1300

240,000

0.04

2-5

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (280 nm)Correction Factor (260 nm)
ReadiLink™ Rapid FITC Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*491516730000.920.35-
ReadiLink™ Rapid Cy5 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*65167025000010.271, 0.420.030.02
ReadiLink™ Rapid Cy7 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*7567792500000.30.0360.05
ReadiLink™ Rapid XFD488 Antibody Labeling Kit *XFD488 Same Structure to Alexa Fluor™ 488*499520710000.9210.110.30
ReadiLink™ Rapid XFD555 Antibody Labeling Kit *XFD555 Same Structure to Alexa Fluor™ 555*5535681500000.110.080.08
ReadiLink™ Rapid XFD594 Antibody Labeling Kit *XFD594 Same Structure to Alexa Fluor™ 594*590618900000.6610.560.43
ReadiLink™ Rapid XFD647 Antibody Labeling Kit *XFD647 Same Structure to Alexa Fluor™ 647*6506712390000.3310.030.00
ReadiLink™ Rapid XFD750 Antibody Labeling Kit *XFD750 Same Structure to Alexa Fluor™ 750*7527762400000.1210.040.00
ReadiLink™ Rapid FITC Antibody Labeling Kit *Production Scale*491516730000.920.35-
ReadiLink™ Rapid Cy5 Antibody Labeling Kit *Production Scale*65167025000010.271, 0.420.030.02
ReadiLink™ Rapid XFD488 Antibody Labeling Kit *Production Scale*499520710000.9210.110.30
ReadiLink™ Rapid XFD555 Antibody Labeling Kit *Production Scale*5535681500000.110.080.08
ReadiLink™ Rapid XFD594 Antibody Labeling Kit *Production Scale*590618900000.6610.560.43
ReadiLink™ Rapid Cy7 Antibody Labeling Kit *Production Scale*7567792500000.30.0360.05
ReadiLink™ Rapid XFD647 Antibody Labeling Kit *Production Scale*6506712390000.3310.030.00
ReadiLink™ Rapid XFD750 Antibody Labeling Kit *Production Scale*7527762400000.1210.040.00
Show More (7)

Citations

View all 1 citations: Citation Explorer
Affinity Peptide-Tethered Suspension Hydrogel Sensor for Selective and Sensitive Detection of Influenza Virus
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)

References

View all 9 references: Citation Explorer
Cy3 Cyanine Dye with Strong Fluorescence Enhancement for AGRO100 and Its Derivative.
Authors: Ma, Xiaoying and Shi, Lei and Zhang, Buyue and Zhao, Shuhua and Yuan, Xinyu and Zhang, Xiufeng
Journal: The journal of physical chemistry. B (2023): 1811-1818
Molecularly Precise, Bright, Photostable, and Biocompatible Cyanine Nanodots as Alternatives to Quantum Dots for Biomedical Applications.
Authors: Yang, Jiajia and Wang, Kaiqi and Zheng, Yihuan and Piao, Ying and Wang, Jinqiang and Tang, Jianbin and Shen, Youqing and Zhou, Zhuxian
Journal: Angewandte Chemie (International ed. in English) (2022): e202202128
Effect of combined VEGF165/ SDF-1 gene therapy on vascular remodeling and blood perfusion in cerebral ischemia.
Authors: Hu, Guo-Jie and Feng, Yu-Gong and Lu, Wen-Peng and Li, Huan-Ting and Xie, Hong-Wei and Li, Shi-Fang
Journal: Journal of neurosurgery (2017): 670-678
Does broodstock nutritional history affect the response of progeny to different first-feeding diets? A whole-body transcriptomic study of rainbow trout alevins.
Authors: Lazzarotto, Viviana and Corraze, Geneviève and Larroquet, Laurence and Mazurais, David and Médale, Françoise
Journal: The British journal of nutrition (2016): 2079-92
Carbon nanotube-based multicolor fluorescent peptide probes for highly sensitive multiplex detection of cancer-related proteases.
Authors: Huang, Yong and Shi, Ming and Hu, Kun and Zhao, Shulin and Lu, Xin and Chen, Zhen-Feng and Chen, Jia and Liang, Hong
Journal: Journal of materials chemistry. B (2013): 3470-3476
Page updated on November 7, 2024

Ordering information

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Unit size
Catalog Number5722
Quantity
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Spectral properties

Correction Factor (260 nm)

0.07

Correction Factor (280 nm)

0.073

Extinction coefficient (cm -1 M -1)

1500001

Excitation (nm)

555

Emission (nm)

569

Quantum yield

0.151

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Components