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AAT Bioquest

mFluor™ UV540 SE

<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; UV540 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; UV540 specific UV9-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; UV540 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; UV540 specific UV9-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; UV540 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; UV540 specific UV9-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.
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Physical properties
Molecular weight1611.98
SolventDMSO
Spectral properties
Absorbance (nm)541
Correction Factor (260 nm)0.634
Correction Factor (280 nm)0.463
Extinction coefficient (cm -1 M -1)900001
Excitation (nm)542
Emission (nm)560
Quantum yield0.351
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
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OverviewpdfSDSpdfProtocol


Molecular weight
1611.98
Absorbance (nm)
541
Correction Factor (260 nm)
0.634
Correction Factor (280 nm)
0.463
Extinction coefficient (cm -1 M -1)
900001
Excitation (nm)
542
Emission (nm)
560
Quantum yield
0.351
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 540 dyes have fluorescence excitation and emission maxima of ~350 nm and ~540 nm respectively. These spectral characteristics make them a unique color for flow cytometry application. mFluor™ UV 540 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. mFluor™ UV 540 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.

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™ UV540 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ UV540 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™ UV540 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™ UV540 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 mM62.036 µL310.178 µL620.355 µL3.102 mL6.204 mL
5 mM12.407 µL62.036 µL124.071 µL620.355 µL1.241 mL
10 mM6.204 µL31.018 µL62.036 µL310.178 µL620.355 µL

Molarity calculator

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Spectrum


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spectrum

Spectral properties

Absorbance (nm)541
Correction Factor (260 nm)0.634
Correction Factor (280 nm)0.463
Extinction coefficient (cm -1 M -1)900001
Excitation (nm)542
Emission (nm)560
Quantum yield0.351

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
mFluor™ UV375 SE3513873000010.9410.0990.138
mFluor™ UV460 SE3584561500010.8610.350.134
mFluor™ UV420 SE353421750001---
mFluor™ UV455 SE3574612000010.4210.6510.406
mFluor™ UV520 SE503524800001-0.4950.518
mFluor™ UV610 SE5906099000010.250.9490.904
mFluor™ UV540 maleimide5425609000010.3510.6340.463

Images


References


View all 2 references: Citation Explorer
Near-ultraviolet laser diodes for brilliant ultraviolet fluorophore excitation.
Authors: Telford, William G
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2015): 1127-37
Nucleic acid dyes for detection of apoptosis in live cells.
Authors: Frey, T
Journal: Cytometry (1995): 265-74