ReadiLink™ Rapid iFluor™ 700 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

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<strong>Readilink™ Kit Labeling Principle: </strong>1).<strong> <em><u>Start</u> </em></strong>the labeling reaction by mixing a labeling dye with a protein (to be labeled) in the Reaction Buffer (pH 7.5-8.5). 2). <strong><em><u>Incubation</u></em> </strong>gives a mixture of the desired protein conjugate and unreactive free dye. 3). <strong><em><u>Quench</u> </em></strong>the reaction by mixing a non-fluorescent Tide Quencher™ (TQ) dye with the reaction solution. The TQ dye stops the reaction AND converts the unreactive free labeling dye to the non-fluorescent TQ-Labeling dye complex, which eliminates the background fluorescence interference of the free labeling dye.
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Unit Size: Cat No: Price (USD): Qty:
2 Labelings 1245 $145


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Superior Labeling Dyes
iFluor Dyes and Kits
Related General proteins
Labeling via Amino Groups
Secondary Reagents
AAT Bioquest's iFluor™ dyes are developed for labeling proteins, in particular, antibodies. These dyes are optimized to have minimal fluorescence quenching effect on proteins and nucleic acids. iFluor™ 700 dyes have fluorescence excitation and emission maxima close to 690 nm and 710 nm respectively. These spectral characteristics make them an excellent alternative to Alexa Fluor® 700 (Alexa Fluor® is the trademark of Invitrogen). ReadiLink™ labeling kits essentially only require 2 simple mixing steps without a column purification needed. iFluor™ 700 SE used in this ReadiLink™ kit is reasonably stable and shows good reactivity and selectivity with protein amino groups. The kit has all the essential components for labeling ~2x50 ug antibody. Each of the two vials of iFluor™ 700 dye provided in the kit is optimized for labeling ~50 µg antibody. iFluor™ 700 SE protein labeling kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor™ 700 SE.




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Protocol


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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

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.

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. Readilink™ Kit Labeling Principle: 1). Start the labeling reaction by mixing a labeling dye with a protein (to be labeled) in the Reaction Buffer (pH 7.5-8.5). 2). Incubation gives a mixture of the desired protein conjugate and unreactive free dye. 3). Quench the reaction by mixing a non-fluorescent Tide Quencher™ (TQ) dye with the reaction solution. The TQ dye stops the reaction AND converts the unreactive free labeling dye to the non-fluorescent TQ-Labeling dye complex, which eliminates the background fluorescence interference of the free labeling dye.
Disclaimer
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 info@aatbio.com if you have any questions.





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References

Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: D Gao, N Liu, Y Li, Y Zhang, G Liu
Journal: Metabolomics (Los Angel) (2017): 2153--0769

Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Kenneth Wu, Robert A Chong, Qing Yu, Jin Bai, Donald E Spratt, Kevin Ching, Chan Lee, Haibin Miao, Inger Tappin, Jerard Hurwitz
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018

Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function
Authors: Helene Piccard, Nele Berghmans, Eva Korpos, Chris Dillen, Ilse Van Aelst, Sandra Li, Erik Martens, Sandra Liekens, Sam Noppen, Jo Van Damme
Journal: International Journal of Cancer (2012): E425--E436