ReadiLink™ Rapid mFluor™ Violet 540 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction

Flow cytometry analysis of HL-60 cells stained with 1ug/ml Mouse IgG control (Green) or with 1ug/ml mouse Anti-Human HLA-ABC (W6/32 mAb)  (Red) and  then followed by Goat Anti-Mouse IgG-mFluor™ Violet 540 conjugate prepared with ReadiLink™ Rapid mFluor™ Violet 540 Antibody Labeling Kit (Cat#1114). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the AmCyan channel.

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Spectral properties

Absorbance (nm)	401
Correction Factor (260 nm)	1.326
Correction Factor (280 nm)	0.543
Extinction coefficient (cm ^-1 M ^-1)	18000¹
Excitation (nm)	402
Emission (nm)	535
Quantum yield	0.21¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

Absorbance (nm)

401

Correction Factor (260 nm)

1.326

Correction Factor (280 nm)

0.543

Extinction coefficient (cm ^-1 M ^-1)

18000¹

Excitation (nm)

402

Emission (nm)

535

Quantum yield

0.21¹

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™ Violet 540 dyes have fluorescence excitation and emission maxima of ~405 nm and ~540 nm respectively. These spectral characteristics make them an excellent replacement for Pacific Orange™ labeling dye. mFluor™ Violet 540 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 purification needed. Each of the two vials of mFluor™ Violet 540 SE provided in the kit is optimized for labeling ~50 µg antibody. mFluor™ Violet 540 SE antibody labeling kit provides a convenient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the Violet Laser-excitable mFluor™ Violet 540 SE.

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, 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

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.
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

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.
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

Spectral properties

Absorbance (nm)	401
Correction Factor (260 nm)	1.326
Correction Factor (280 nm)	0.543
Extinction coefficient (cm ^-1 M ^-1)	18000¹
Excitation (nm)	402
Emission (nm)	535
Quantum yield	0.21¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	406	445	35000¹	0.81¹	0.338	0.078
ReadiLink™ Rapid mFluor™ Violet 420 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	403	427	37000¹	0.91¹	-	-
ReadiLink™ Rapid mFluor™ Violet 510 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	412	505	25000¹	0.86¹	0.464	0.366

Images

Figure 1. Flow cytometry analysis of HL-60 cells stained with 1ug/ml Mouse IgG control (Green) or with 1ug/ml mouse Anti-Human HLA-ABC (W6/32 mAb) (Red) and then followed by Goat Anti-Mouse IgG-mFluor™ Violet 540 conjugate prepared with ReadiLink™ Rapid mFluor™ Violet 540 Antibody Labeling Kit (Cat#1114). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the AmCyan channel.

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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