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mFluor™ UV520 SE

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.
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 Number1643
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 weight1531.85
SolventDMSO
Spectral properties
Absorbance (nm)502
Correction Factor (260 nm)0.495
Correction Factor (280 nm)0.518
Extinction coefficient (cm -1 M -1)800001
Excitation (nm)503
Emission (nm)524
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
1531.85
Absorbance (nm)
502
Correction Factor (260 nm)
0.495
Correction Factor (280 nm)
0.518
Extinction coefficient (cm -1 M -1)
800001
Excitation (nm)
503
Emission (nm)
524
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 520 dyes have fluorescence excitation and emission maxima of ~350 nm and ~520 nm respectively. These spectral characteristics make them a unique color for flow cytometry application. mFluor™ UV 520 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. mFluor™ UV 520 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™ UV520 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ UV520 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™ UV520 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™ UV520 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 mM65.281 µL326.403 µL652.805 µL3.264 mL6.528 mL
5 mM13.056 µL65.281 µL130.561 µL652.805 µL1.306 mL
10 mM6.528 µL32.64 µL65.281 µL326.403 µL652.805 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)502
Correction Factor (260 nm)0.495
Correction Factor (280 nm)0.518
Extinction coefficient (cm -1 M -1)800001
Excitation (nm)503
Emission (nm)524

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™ UV540 SE5425609000010.3510.6340.463
mFluor™ UV610 SE5906099000010.250.9490.904

References


View all 28 references: Citation Explorer
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Authors: Chayé, Mathilde A M and Tontini, Chiara and Ozir-Fazalalikhan, Arifa and Voskamp, Astrid L and Smits, Hermelijn H
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 235-261
Distinct cellular immune profiles in the airways and blood of critically ill patients with COVID-19.
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Journal: The Journal of allergy and clinical immunology (2021)
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Journal: Journal of immunology (Baltimore, Md. : 1950) (2021): 206-213
Human Lung-Resident Macrophages Colocalize with and Provide Costimulation to PD1hi Tissue-Resident Memory T Cells.
Authors: Snyder, Mark E and Sembrat, John and Noda, Kentaro and Myerburg, Michael M and Craig, Andrew and Mitash, Nilay and Harano, Takashi and Furukawa, Masashi and Pilewski, Joseph and McDyer, John and Rojas, Mauricio and Sanchez, Pablo
Journal: American journal of respiratory and critical care medicine (2021): 1230-1244
A Comprehensive Workflow for Applying Single-Cell Clustering and Pseudotime Analysis to Flow Cytometry Data.
Authors: Melsen, Janine E and van Ostaijen-Ten Dam, Monique M and Lankester, Arjan C and Schilham, Marco W and van den Akker, Erik B
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Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)
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Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)
Full Spectrum Flow Cytometry as a Powerful Technology for Cancer Immunotherapy Research.
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