mFluor™ UV420 SE
Ordering information
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Physical properties
Molecular weight | 1385.78 |
Solvent | DMSO |
Spectral properties
Absorbance (nm) | 352 |
Extinction coefficient (cm -1 M -1) | 750001 |
Excitation (nm) | 353 |
Emission (nm) | 421 |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12171501 |
Overview | ![]() ![]() |
See also: Amine Reactive Dyes and Probes for Conjugation, mFluor™ Dyes and Kits, Spectral Flow Cytometry
Molecular weight 1385.78 | Absorbance (nm) 352 | Extinction coefficient (cm -1 M -1) 750001 | Excitation (nm) 353 | Emission (nm) 421 |
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., 350 nm, 405 nm, 488 nm and 633 nm). mFluor™ UV dyes are optimized to be excited with a UV laser at 350 nm. AAT Bioquest offers the largest collection of fluorescent dyes that are excited by UV laser at 350 nm. mFluor™ UV 420 dyes have fluorescence excitation and emission maxima of ~350 nm and ~420 nm respectively. These spectral characteristics make them a unique color for flow cytometry application. mFluor™ UV 420 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. mFluor™ UV 420 SE provides a convenient tool to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) for flow cytometric applications with the UV 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.
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.
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.
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™ UV420 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ UV420 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™ UV420 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.
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
- 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. - 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.- Prepare Sephadex G-25 column according to the manufacture instruction.
- Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
- Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
- 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™ UV420 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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 72.162 µL | 360.808 µL | 721.615 µL | 3.608 mL | 7.216 mL |
5 mM | 14.432 µL | 72.162 µL | 144.323 µL | 721.615 µL | 1.443 mL |
10 mM | 7.216 µL | 36.081 µL | 72.162 µL | 360.808 µL | 721.615 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
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Spectrum
Open in Advanced Spectrum Viewer


Spectral properties
Absorbance (nm) | 352 |
Extinction coefficient (cm -1 M -1) | 750001 |
Excitation (nm) | 353 |
Emission (nm) | 421 |
Product Family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
mFluor™ UV375 SE | 351 | 387 | 300001 | 0.941 | 0.099 | 0.138 |
mFluor™ UV460 SE | 358 | 456 | 150001 | 0.861 | 0.35 | 0.134 |
mFluor™ UV455 SE | 357 | 461 | 200001 | 0.421 | 0.651 | 0.406 |
mFluor™ UV520 SE | 503 | 524 | 800001 | - | 0.495 | 0.518 |
mFluor™ UV540 SE | 542 | 560 | 900001 | 0.351 | 0.634 | 0.463 |
mFluor™ UV610 SE | 590 | 609 | 900001 | 0.25 | 0.949 | 0.904 |
Images

Figure 1. Top) 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. Bottom) Flow cytometry analysis of whole blood cells stained with CD4-mFluor™ UV420 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ UV420 specific UV3-A channel.

Figure 2. 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-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.
References
View all 1 references: Citation Explorer
Influenza-specific IgG1+ memory B-cell numbers increase upon booster vaccination in healthy adults but not in patients with predominantly antibody deficiency.
Authors: Hartley, Gemma E and Edwards, Emily S J and Bosco, Julian J and Ojaimi, Samar and Stirling, Robert G and Cameron, Paul U and Flanagan, Katie and Plebanski, Magdalena and Hogarth, Philip Mark and O'Hehir, Robyn E and van Zelm, Menno C
Journal: Clinical & translational immunology (2020): e1199
Authors: Hartley, Gemma E and Edwards, Emily S J and Bosco, Julian J and Ojaimi, Samar and Stirling, Robert G and Cameron, Paul U and Flanagan, Katie and Plebanski, Magdalena and Hogarth, Philip Mark and O'Hehir, Robyn E and van Zelm, Menno C
Journal: Clinical & translational immunology (2020): e1199
Application notes
A New Protein Crosslinking Method for Labeling and Modifying Antibodies
Abbreviation of Common Chemical Compounds Related to Peptides
Bright Tide Fluor™-Based Fluorescent Peptides and Their Applications In Drug Discovery and Disease Diagnosis
FITC (Fluorescein isothiocyanate)
Fluorescein isothiocyanate (FITC)
Abbreviation of Common Chemical Compounds Related to Peptides
Bright Tide Fluor™-Based Fluorescent Peptides and Their Applications In Drug Discovery and Disease Diagnosis
FITC (Fluorescein isothiocyanate)
Fluorescein isothiocyanate (FITC)