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AAT Bioquest

ReadiLink™ Rapid mFluor™ Blue 570 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*

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™ Blue 570 dyes have fluorescence excitation and emission maxima of ~553 nm and ~570 nm respectively. Like RPE protein, it has a broad absorption spectrum, making its conjugates readily excitable by 488 nm blue laser. These spectral characteristics make mFluor™ Blue 570 an excellent alternative to RPE labeling protein. mFluor™ Blue 570 is a small organic molecule that is much easier to use than the RPE labeling protein. mFluor™ Blue 570 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™ Blue 570 provided in the kit is optimized for labeling ~50 µg antibody. mFluor™ Blue 570 antibody labeling kit provides a convenient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the mFluor™ Blue 570 SE 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.

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

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

View all 59 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 September 18, 2024

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Catalog Number1120
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Spectral properties

Absorbance (nm)

553

Correction Factor (260 nm)

0.228

Correction Factor (280 nm)

0.179

Extinction coefficient (cm -1 M -1)

1200001

Excitation (nm)

505

Emission (nm)

564

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 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™ Blue 570 Antibody Labeling Kit (Cat No. 1120). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the PE 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™ Blue 570 Antibody Labeling Kit (Cat No. 1120). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the PE 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™ Blue 570 Antibody Labeling Kit (Cat No. 1120). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the PE channel.