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

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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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2 Labelings 1292 $145

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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
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Category Superior Labeling Dyes
iFluor Dyes and Kits
Related General proteins
Labeling via Amino Groups
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).

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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Table 1. Available fluorophores in AAT Bioquest ReadyLink™ Rapid Antibody Labelling Kits

Cat# Labels Ex (nm) Em (nm)
1100  mFluor™ Violet 450 403 454
1105 mFluor™ Violet 420 398 411
1110 mFluor™ Violet 510  414  508
1114 mFluor™ Violet 540  399  550
1120 mFluor™ Blue 570 553  570 
1123 mFluor™ Green 620  522  617
1126 mFluor™ Yellow 630  561  630
1130 mFluor™ Red 700   657 700 
1131  mFluor™ Red 780 629  780 
1220 iFluor™ 350   345 442
1227 iFluor™ 555  559  569
1230  iFluor™ 594  592 614
1235  iFluor™ 647  654 674
1240  iFluor™ 680  682 701
1245  iFluor™ 700  693 713
1250  iFluor™ 750  753 779
1255  iFluor™ 488  491 514
1260  iFluor™ 633  638 655
1265 iFluor™ 790   782 811
1290  Cy3  555 565
1292  Cy5  644 665
1294  Cy7  749  776
1299  FITC 494  520
Preparation of working solution


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.

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 

  1. 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.

  2. 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

  1. 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.

  2. 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.

Example data analysis and figures

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).
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email if you have any questions.


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