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.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Physical properties

Molecular weight	1531.85
Solvent	DMSO

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-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 SDS Protocol

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

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™ 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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	65.281 µL	326.403 µL	652.805 µL	3.264 mL	6.528 mL
5 mM	13.056 µL	65.281 µL	130.561 µL	652.805 µL	1.306 mL
10 mM	6.528 µL	32.64 µL	65.281 µL	326.403 µL	652.805 µL

Molarity calculator

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Spectrum

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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

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™ UV420 SE	353	421	75000¹	-	-	-
mFluor™ UV455 SE	357	461	20000¹	0.42¹	0.651	0.406
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. 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 28 references: Citation Explorer

Use of Toll-Like Receptor (TLR) Ligation to Characterize Human Regulatory B-Cells Subsets.
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.
Authors: Saris, Anno and Reijnders, Tom Dy and Nossent, Esther J and Schuurman, Alex R and Verhoeff, Jan and Asten, Saskia van and Bontkes, Hetty and Blok, Siebe and Duitman, Janwillem and Bogaard, Harm-Jan and Heunks, Leo and Lutter, Rene and van der Poll, Tom and Garcia Vallejo, Juan J and ,
Journal: Thorax (2021)

Basophils promote barrier dysfunction and resolution in the atopic skin.
Authors: Pellefigues, Christophe and Naidoo, Karmella and Mehta, Palak and Schmidt, Alfonso J and Jagot, Ferdinand and Roussel, Elsa and Cait, Alissa and Yumnam, Bibek and Chappell, Sally and Meijlink, Kimberley and Camberis, Mali and Jiang, Jean X and Painter, Gavin and Filbey, Kara and Uluçkan, Özge and Gasser, Olivier and Le Gros, Graham
Journal: The Journal of allergy and clinical immunology (2021)

Multibatch Cytometry Data Integration for Optimal Immunophenotyping.
Authors: Ogishi, Masato and Yang, Rui and Gruber, Conor and Zhang, Peng and Pelham, Simon J and Spaan, András N and Rosain, Jérémie and Chbihi, Marwa and Han, Ji Eun and Rao, V Koneti and Kainulainen, Leena and Bustamante, Jacinta and Boisson, Bertrand and Bogunovic, Dusan and Boisson-Dupuis, Stéphanie and Casanova, Jean-Laurent
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
Journal: Journal of immunology (Baltimore, Md. : 1950) (2020)

High-Dimensional Data Analysis Algorithms Yield Comparable Results for Mass Cytometry and Spectral Flow Cytometry Data.
Authors: Ferrer-Font, Laura and Mayer, Johannes U and Old, Samuel and Hermans, Ian F and Irish, Jonathan and Price, Kylie M
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

HTLV infected individuals have increased B-cell activation and proinflammatory regulatory T-cells.
Authors: Kjerulff, Bertram and Petersen, Mikkel Steen and Rodrigues, Candida Medina and da Silva Té, David and Christiansen, Mette and Erikstrup, Christian and Hønge, Bo Langhoff
Journal: Immunobiology (2020): 151878

Multispectral Flow Cytometry: Unaddressed Issues and Recommendations for Improvement.
Authors: Parks, David R
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.
Authors: Bonilla, Diana L and Reinin, Gil and Chua, Edmond
Journal: Frontiers in molecular biosciences (2020): 612801