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AAT Bioquest

mFluor™ Violet 510 SE

AAT Bioquest's mFluor™ dyes are developed for multicolor 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 510 dyes have fluorescence excitation and emission maxima of ~405 nm and ~510 nm respectively. These spectral characteristics make them an excellent replacement for Pacific Orange™ labeling dye. mFluor™ Violet 510 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. mFluor™ Violet 510 SE provides a convenient tool to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) for flow cytometric applications with the violet laser excitation.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 1 M  sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution.
Note     The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M  sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
Note     The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, 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. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
Note     The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.


2. mFluor™ Violet 510 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ Violet 510 SE to make a 10 mM stock solution. Mix well by pipetting or vortex.
Note     Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with mFluor™ Violet 510 SE. You might need further optimization for your particular proteins.
Note     Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.


Run conjugation reaction
  1. Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point:  Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.
    Note     We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
  2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes. 

Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.
  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.
    Note     For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses.
    Note     For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried. 

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of mFluor™ Violet 510 SE to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM129.73 µL648.651 µL1.297 mL6.487 mL12.973 mL
5 mM25.946 µL129.73 µL259.461 µL1.297 mL2.595 mL
10 mM12.973 µL64.865 µL129.73 µL648.651 µL1.297 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
mFluor™ Violet 450 SE4064453500010.8110.3380.078
mFluor™ Violet 540 SE4025351800010.2111.3260.543
mFluor™ Violet 510 acid4125052500010.8610.4640.366
mFluor™ Violet 500 SE4105012500010.8110.7690.365
mFluor™ Violet 610 SE5946129000010.310.5320.66
mFluor™ Violet 550 SE5275509000010.3110.4740.306
mFluor™ Violet 505 SE3935044000010.4510.8880.403
mFluor™ Violet 590 SE5645919000010.2210.6320.329
mFluor™ Violet 545 SE3935432000010.1511.080.496
mFluor™ Violet 530 SE393543200001---
mFluor™ Violet 480 SE40447540,000-1.230.6570
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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 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 September 19, 2024

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Catalog Number1151
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Physical properties

Molecular weight

770.83

Solvent

DMSO

Spectral properties

Absorbance (nm)

410

Correction Factor (260 nm)

0.464

Correction Factor (280 nm)

0.366

Extinction coefficient (cm -1 M -1)

250001

Excitation (nm)

412

Emission (nm)

505

Quantum yield

0.861

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501
<strong>Top)</strong>&nbsp;Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature.&nbsp;<strong>Bottom)</strong>&nbsp;Flow cytometry analysis of whole blood cells stained with CD4-mFluor&trade; Violet 510 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; Violet 510 specific V5-A channel.
<strong>Top)</strong>&nbsp;Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature.&nbsp;<strong>Bottom)</strong>&nbsp;Flow cytometry analysis of whole blood cells stained with CD4-mFluor&trade; Violet 510 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; Violet 510 specific V5-A channel.
<strong>Top)</strong>&nbsp;Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature.&nbsp;<strong>Bottom)</strong>&nbsp;Flow cytometry analysis of whole blood cells stained with CD4-mFluor&trade; Violet 510 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; Violet 510 specific V5-A channel.
Signal-to-noise ratio of flow cytometric analysis of CD45 expression on human lymphocytes. Whole blood was stained with Mouse Anti-Human CD45 conjugated with mFluor&trade; Violet 510 (Cat#1151) or Pacific Orange with different dye-to-protein ratio (D/P). mFluor&trade; Violet 510 offers improved brightness over Pacific Orange.