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AAT Bioquest

ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*

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. Our iFluor® 750 dyes have fluorescence excitation and emission maxima close to 750 nm and 780 nm respectively with good photostability. Our in-house comparable studies indicated that our iFluor® 750 dyes are significantly brighter than the corresponding Cy7® and Alexa Fluor® 750. These spectral characteristics make them a superior alternative to Cy7® and Alexa Fluor® 750 (Cy7® and Alexa Fluor® are the trademarks of GE Healthcare and Invitrogen respectively). iFluor® 750 conjugates have been widely used in fluorescence animal imaging applications. ReadiLink™ labeling kits essentially only require 2 simple mixing steps without a column purification needed. iFluor® 750 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® 750 dye provided in the kit is optimized for labeling ~50 µg antibody. iFluor® 750 SE protein labeling kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor® 750 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.

Example protocol

AT A GLANCE

Important

Warm all the components and centrifuge the vials briefly before opening them. Immediately prepare the necessary solutions before starting your conjugation. The following protocol is a 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: A final protein concentration range of 1 - 2 mg/mL is recommended for optimal labeling efficiency, 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 a 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

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
ReadiLink™ Rapid iFluor® 350 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*3454502000010.9510.830.23
ReadiLink™ Rapid iFluor® 555 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*55757010000010.6410.230.14
ReadiLink™ Rapid iFluor® 594 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*58760320000010.5310.050.04
ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*65667025000010.2510.030.03
ReadiLink™ Rapid iFluor® 680 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*68470122000010.2310.0970.094
ReadiLink™ Rapid iFluor® 700 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*69071322000010.2310.090.04
ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*4915167500010.910.210.11
ReadiLink™ Rapid iFluor® 633 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*64065425000010.2910.0620.044
ReadiLink™ Rapid iFluor® 790 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*78781225000010.1310.10.09
ReadiLink™ Rapid iFluor® 350 Antibody Labeling Kit *Production Scale*3454502000010.9510.830.23
ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit *Production Scale*4915167500010.910.210.11
ReadiLink™ Rapid iFluor® 555 Antibody Labeling Kit *Production Scale*55757010000010.6410.230.14
ReadiLink™ Rapid iFluor® 594 Antibody Labeling Kit *Production Scale*58760320000010.5310.050.04
ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit *Production Scale*65667025000010.2510.030.03
Show More (5)

Citations

View all 4 citations: Citation Explorer
Secretions from hypochlorous acid-treated tumor cells delivered in a melittin hydrogel potentiate cancer immunotherapy
Authors: Zhou, Yuhan and Ye, Ting and Ye, Chengzhi and Wan, Chao and Yuan, Siyue and Liu, Yushuai and Li, Tianyu and Jiang, Fagang and Lovell, Jonathan F and Jin, Honglin and others,
Journal: Bioactive Materials (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
Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
Journal: Mol Biosyst (2011): 1766
Visualizing dengue virus through Alexa Fluor labeling
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)
Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells
Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
Journal: Biochem Biophys Res Commun (2011): 211
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
Authors: Cui JJ, Zhu XL, Ji CF, Jing XH, Bai WZ.
Journal: Zhen Ci Yan Jiu (2011): 262
Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
Authors: Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S.
Journal: J Microbiol Methods (2011): 119
Page updated on October 12, 2024

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Catalog Number1250
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Spectral properties

Correction Factor (260 nm)

0.044

Correction Factor (280 nm)

0.039

Correction Factor (565 nm)

0.0250

Correction Factor (650 nm)

0.1413

Extinction coefficient (cm -1 M -1)

2750001

Excitation (nm)

757

Emission (nm)

779

Quantum yield

0.121

Storage, safety and handling

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

Components

HL-60 cells were incubated with (red) or without (green) anti-human HLA-ABC (W6/32 mAb). Cells were then incubated with goat anti-mouse IgG labeled using the ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit (Cat No. 1250). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the APC-Cy7 channel.
HL-60 cells were incubated with (red) or without (green) anti-human HLA-ABC (W6/32 mAb). Cells were then incubated with goat anti-mouse IgG labeled using the ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit (Cat No. 1250). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the APC-Cy7 channel.
HL-60 cells were incubated with (red) or without (green) anti-human HLA-ABC (W6/32 mAb). Cells were then incubated with goat anti-mouse IgG labeled using the ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit (Cat No. 1250). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the APC-Cy7 channel.