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mFluor™ Violet 590 SE

<strong>Top)</strong> 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. <strong>Bottom)</strong> Flow cytometry analysis of whole blood cells stained with CD4-mFluor™ Violet 590 conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the mFluor™ Violet 590 specific V9-A channel.
<strong>Top)</strong> 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. <strong>Bottom)</strong> Flow cytometry analysis of whole blood cells stained with CD4-mFluor™ Violet 590 conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the mFluor™ Violet 590 specific V9-A channel.
Fluorescent dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating dyes to a peptide, protein, antibody, amino-modified oligonucleotide, or nucleic acid. NHS esters react readily with the primary amines (R-NH<sub>2</sub>) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.
Ordering information
Price ()
Catalog Number1155
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight1277.89
SolventDMSO
Spectral properties
Absorbance (nm)563
Correction Factor (260 nm)0.632
Correction Factor (280 nm)0.329
Extinction coefficient (cm -1 M -1)900001
Excitation (nm)564
Emission (nm)591
Quantum yield0.221
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
1277.89
Absorbance (nm)
563
Correction Factor (260 nm)
0.632
Correction Factor (280 nm)
0.329
Extinction coefficient (cm -1 M -1)
900001
Excitation (nm)
564
Emission (nm)
591
Quantum yield
0.221
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 dyes are optimized to be excited with a violet laser at 405 nm. AAT Bioquest offers the largest collection of fluorescent dyes that are excited by violet laser. mFluor™ Violet 590 dyes have fluorescence excitation and emission maxima of ~405 nm and ~590 nm respectively. These spectral characteristics make them a unique color for flow cytometry application. mFluor™ Violet 590 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. mFluor™ Violet 590 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 590 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ Violet 590 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 590 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 590 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 mM78.254 µL391.27 µL782.54 µL3.913 mL7.825 mL
5 mM15.651 µL78.254 µL156.508 µL782.54 µL1.565 mL
10 mM7.825 µL39.127 µL78.254 µL391.27 µL782.54 µL

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


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spectrum

Spectral properties

Absorbance (nm)563
Correction Factor (260 nm)0.632
Correction Factor (280 nm)0.329
Extinction coefficient (cm -1 M -1)900001
Excitation (nm)564
Emission (nm)591
Quantum yield0.221

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 510 SE4125052500010.8610.4640.366
mFluor™ Violet 540 SE4025351800010.2111.3260.543
mFluor™ Violet 500 SE4105012500010.8110.7690.365
mFluor™ Violet 610 SE4216133500010.310.5320.66
mFluor™ Blue 590 SE5695898100010.1510.6710.406
mFluor™ Violet 550 SE5275509000010.3110.4740.306
mFluor™ Violet 505 SE3935044000010.4510.8880.403
mFluor™ Violet 545 SE3935432000010.1511.080.496
mFluor™ Violet 530 SE393543200001---
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References


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