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

HL-60 cells were stained with (Red) or without (Green) 1 µg/mL anti-human HLA-ABC-Biotin (W6/32 mAb). Cells were then incubated with streptavidin labeled using the ReadiLink™ Rapid mFluor™ Red 700 Antibody Labeling Kit (Cat No. 1130). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the APC channel.
HL-60 cells were stained with (Red) or without (Green) 1 µg/mL anti-human HLA-ABC-Biotin (W6/32 mAb). Cells were then incubated with streptavidin labeled using the ReadiLink™ Rapid mFluor™ Red 700 Antibody Labeling Kit (Cat No. 1130). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the APC channel.
HL-60 cells were stained with (Red) or without (Green) 1 µg/mL anti-human HLA-ABC-Biotin (W6/32 mAb). Cells were then incubated with streptavidin labeled using the ReadiLink™ Rapid mFluor™ Red 700 Antibody Labeling Kit (Cat No. 1130). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the APC channel.
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Spectral properties
Absorbance (nm)651
Correction Factor (260 nm)0.135
Correction Factor (280 nm)0.127
Extinction coefficient (cm -1 M -1)2950001
Excitation (nm)680
Emission (nm)695
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Absorbance (nm)
651
Correction Factor (260 nm)
0.135
Correction Factor (280 nm)
0.127
Extinction coefficient (cm -1 M -1)
2950001
Excitation (nm)
680
Emission (nm)
695
AAT Bioquest's mFluor™ dyes are developed for flow cytometry-focused applications. These dyes have large Stokes Shifts, and can be well excited by the laser lines of flow cytometers (e.g., 405 nm, 488 nm and 633 nm). mFluor™ Red 700 dyes have fluorescence excitation and emission maxima of ~657 nm and ~700 nm respectively. Like Alexa Fluor 700-APC tandem, it has strong absorption at 633 nm, making its conjugates readily excitable by 633 nm red laser. These spectral characteristics make mFluor™ Red 700 an excellent alternative to Alexa Fluor 700-APC tandem. mFluor™ Red 700 is a small organic molecule that is much easier to use than the Alexa Fluor 700-APC tandem. mFluor™ Red 700 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. This kit has all the essential components to perform two separate labeling reactions with no column purification needed. Each of the two vials of mFluor™ Red 700 provided in the kit is optimized for labeling ~50 µg antibody. mFluor™ Red 700 antibody labeling kit provides a convenient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the mFluor™ Red 700 dye.

 

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


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


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spectrum

Spectral properties

Absorbance (nm)651
Correction Factor (260 nm)0.135
Correction Factor (280 nm)0.127
Extinction coefficient (cm -1 M -1)2950001
Excitation (nm)680
Emission (nm)695

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (260 nm)Correction Factor (280 nm)
ReadiLink™ Rapid mFluor™ Red 780 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*6297679000010.1010.116

Images


Citations


View all 3 citations: Citation Explorer
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


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