mFluor™ Blue 580 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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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	832.98
Solvent	DMSO

Spectral properties

Absorbance (nm)	492
Correction Factor (260 nm)	0.363
Correction Factor (280 nm)	0.247
Extinction coefficient (cm ^-1 M ^-1)	40000¹
Excitation (nm)	485
Emission (nm)	580

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

832.98

Absorbance (nm)

492

Correction Factor (260 nm)

0.363

Correction Factor (280 nm)

0.247

Extinction coefficient (cm ^-1 M ^-1)

40000¹

Excitation (nm)

485

Emission (nm)

580

Advances in spectral flow cytometers have expanded applications and capabilities beyond conventional flow cytometry. Now with spectral flow cytometry analysis, researchers and scientists can investigate an increasing number of molecules of interest. However, the potential of spectral flow cytometry is severely limited by the availability of fluorescent labels and readouts. AAT Bioquest's mFluor™ dyes are developed for multicolor flow cytometry-focused applications, in particular, for spectral fluorescence flow cytometry. mFluor™ Blue 580 dye can be well excited with blue laser at 488 nm. It has a huge Stokes shift with emission ~580 nm. mFluor™ Blue 580 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Blue 580 dyes are well excited at 488 nm to give red fluorescence. mFluor™ Blue 580 dye and conjugates are excellent blue laser reagents for flow cytometry detections. Compared to RPE, mFluor™ Blue 580 dyes are much more photostable, making them readily available for fluorescence imaging applications while it is very difficult to use RPE conjugates for fluorescence imaging applications due to the rapid photobleaching of RPE conjugates. It is also a unique fluorochrome for spectral flow cytometry since there are very few existing dyes that have this spectral profile.

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™ Blue 580 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ Blue 580 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™ Blue 580 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™ Blue 580 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	120.051 µL	600.255 µL	1.201 mL	6.003 mL	12.005 mL
5 mM	24.01 µL	120.051 µL	240.102 µL	1.201 mL	2.401 mL
10 mM	12.005 µL	60.025 µL	120.051 µL	600.255 µL	1.201 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Absorbance (nm)	492
Correction Factor (260 nm)	0.363
Correction Factor (280 nm)	0.247
Extinction coefficient (cm ^-1 M ^-1)	40000¹
Excitation (nm)	485
Emission (nm)	580

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Correction Factor (260 nm)	Correction Factor (280 nm)
mFluor™ Blue 570 SE	553	565	120000¹	0.228	0.179
mFluor™ Blue 630 SE	470	634	49000¹	0.197	0.275
mFluor™ Blue 660 SE	481	663	26000¹	0.338	0.32
mFluor™ Blue 590 SE	569	589	81000¹	0.671	0.406
mFluor™ Blue 620 SE	589	616	98000¹	0.683	0.849

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 50 references: Citation Explorer

Predicting Outcomes of Rat Vascularized Composite Allotransplants through Quantitative Measurement of Chimerism with PCR-Amplified Short Tandem Repeat.
Authors: Cheng, Hui-Yun and Huang, Xiao-Ting and Lin, Chih-Fan and Al Deek, Nidal F and Shih, Ling-Yi and Lin, Cheng-Hung and Wei, Fu-Chan
Journal: Journal of immunology research (2020): 9243531

Worry and FRET: ROS Production Leads to Fluorochrome Tandem Degradation and impairs Interpretation of Flow Cytometric Results.
Authors: Jensen, Isaac J and McGonagill, Patrick W and Lefebvre, Mitchell N and Griffith, Thomas S and Harty, John T and Badovinac, Vladimir P
Journal: Immunity (2020): 419-421

A new tandem peptide modified liposomal doxorubicin for tumor "ecological therapy".
Authors: Zhao, Ting and Zhou, Hongli and Lei, Lei and Guo, Chenqi and Yang, Qin and Gong, Ting and Sun, Xun and Song, Xu and Gong, Tao and Zhang, Zhirong
Journal: Nanoscale (2020): 3359-3369

The Dark Matter of Large Cereal Genomes: Long Tandem Repeats.
Authors: Kapustová, Veronika and Tulpová, Zuzana and Toegelová, Helena and Novák, Petr and Macas, Jiří and Karafiátová, Miroslava and Hřibová, Eva and Doležel, Jaroslav and Šimková, Hana
Journal: International journal of molecular sciences (2019)

Long-term follow up of tandem autologous-allogeneic hematopoietic cell transplantation for multiple myeloma.
Authors: Maffini, Enrico and Storer, Barry E and Sandmaier, Brenda M and Bruno, Benedetto and Sahebi, Firoozeh and Shizuru, Judith A and Chauncey, Thomas R and Hari, Parameswaran and Lange, Thoralf and Pulsipher, Michael A and McSweeney, Peter A and Holmberg, Leona and Becker, Pamela S and Green, Damian J and Mielcarek, Marco and Maloney, David G and Storb, Rainer
Journal: Haematologica (2019): 380-391

Tandem Payne/Dakin Reaction: A New Strategy for Hydrogen Peroxide Detection and Molecular Imaging.
Authors: Ye, Sen and Hu, Jun Jacob and Yang, Dan
Journal: Angewandte Chemie (International ed. in English) (2018): 10173-10177

FIR haplodeficiency promotes splicing to pyruvate kinase M2 in mice thymic lymphoma tissues revealed by six-plex tandem mass tag quantitative proteomic analysis.
Authors: Kimura, Asako and Kitamura, Kouichi and Ailiken, Guzhanuer and Satoh, Mamoru and Minamoto, Toshinari and Tanaka, Nobuko and Nomura, Fumio and Matsushita, Kazuyuki
Journal: Oncotarget (2017): 67955-67965

Mechanism of tandem duplication formation in BRCA1-mutant cells.
Authors: Willis, Nicholas A and Frock, Richard L and Menghi, Francesca and Duffey, Erin E and Panday, Arvind and Camacho, Virginia and Hasty, E Paul and Liu, Edison T and Alt, Frederick W and Scully, Ralph
Journal: Nature (2017): 590-595

Flow cytometry remission by Ig light chains ratio is a powerful marker of outcome in multiple myeloma after tandem autologous transplant: a real-life study.
Authors: Cordone, Iole and Marchesi, Francesco and Masi, Serena and Summa, Valentina and Pisani, Francesco and Merola, Roberta and Cigliana, Giovanni and Orlandi, Giulia and Gumenyuk, Svitlana and Palombi, Francesca and Romano, Atelda and Spadea, Antonio and Renzi, Daniela and Papa, Elena and Canfora, Marco and Conti, Laura and Petti, Maria Concetta and Mengarelli, Andrea
Journal: Journal of experimental & clinical cancer research : CR (2016): 49

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.
Authors: Bigenzahn, Johannes W and Fauster, Astrid and Rebsamen, Manuele and Kandasamy, Richard K and Scorzoni, Stefania and Vladimer, Gregory I and Müller, André C and Gstaiger, Matthias and Zuber, Johannes and Bennett, Keiryn L and Superti-Furga, Giulio
Journal: Molecular & cellular proteomics : MCP (2016): 1139-50