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ReadiLink™ Rapid iFluor® 594 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

HeLa cells were stained with (Tubulin+) or without (Tubulin-) mouse anti-tubulin and then visualized with iFluor<sup>™</sup> 594 goat anti-mouse IgG (Left) or with Alexa Fluor<sup>®</sup> 594 goat anti-mouse IgG (Right).
HeLa cells were stained with (Tubulin+) or without (Tubulin-) mouse anti-tubulin and then visualized with iFluor<sup>™</sup> 594 goat anti-mouse IgG (Left) or with Alexa Fluor<sup>®</sup> 594 goat anti-mouse IgG (Right).
Ordering information
Price ()
Catalog Number1230
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Correction Factor (260 nm)0.05
Correction Factor (280 nm)0.04
Extinction coefficient (cm -1 M -1)1800001
Excitation (nm)588
Emission (nm)604
Quantum yield0.531
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
0.05
Correction Factor (280 nm)
0.04
Extinction coefficient (cm -1 M -1)
1800001
Excitation (nm)
588
Emission (nm)
604
Quantum yield
0.531
AAT Bioquest's iFluor® dyes are optimized for labeling proteins, in particular, antibodies. iFluor® 594 dyes have fluorescence excitation and emission maxima of ~590 nm and ~610 nm respectively. iFluor® 594 family has the spectral properties similar to those of Texas Red® and Alexa Fluor® 594 (Texas Red® and Alexa Fluor® 594 are the trademarks of Invitrogen). iFluor® 594 family is pH-independent from pH 3 to 11. These spectral characteristics make this new dye family an excellent alternative to Texas Red® and Alexa Fluor® 594.Compared to Texas Red®, iFluor® 594 is much easier to be conjugated with RPE with much higher conjugation yield, and the resulted RPE-iFluor® 594 tandem has better FRET efficiency.ReadiLink™ labeling kits essentially only require 2 simple mixing steps without a column purification needed. iFluor® 594 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® 594 dye provided in the kit is optimized for labeling ~50 µg antibody. iFluor® 594 SE protein labeling kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor® 594 SE.

 

readilinkworkflow

 

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


Component A: iFluor™ 5942 vials (One vial is for 50 μg protein)
Component B: Reaction Buffer1 vial (20 μL)
Component C: TQ™-Dyed Quench Buffer1 vial (20 μL)

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
  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.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.05
Correction Factor (280 nm)0.04
Extinction coefficient (cm -1 M -1)1800001
Excitation (nm)588
Emission (nm)604
Quantum yield0.531

Citations


View all 4 citations: Citation Explorer
IBEX: An open and extensible method for high content multiplex imaging of diverse tissues
Authors: Radtke, Andrea J and Chu, Colin J and Yaniv, Ziv and Yao, Li and Marr, James and Beuschel, Rebecca T and Ichise, Hiroshi and Gola, Anita and Kabat, Juraj and Lowekamp, Bradley and others,
Journal: arXiv preprint arXiv:2107.11364 (2021)
Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769
Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018
Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function
Authors: Piccard, Helene and Berghmans, Nele and Korpos, Eva and Dillen, Chris and Aelst, Ilse Van and Li, S and ra , undefined and Martens, Erik and Liekens, S and ra , undefined and Noppen, Sam and Damme, Jo Van and others, undefined
Journal: International Journal of Cancer (2012): E425--E436

References


View all 49 references: Citation Explorer
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Visualizing dengue virus through Alexa Fluor labeling
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Neuroanatomical basis of clinical joint application of "Jinggu" (BL 64, a source-acupoint) and "Dazhong" (KI 4, a Luo-acupoint) in the rat: a double-labeling study of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594
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