mFluor™ Blue 590 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	1783.14
Solvent	DMSO

Spectral properties

Absorbance (nm)	573
Correction Factor (260 nm)	0.671
Correction Factor (280 nm)	0.406
Extinction coefficient (cm ^-1 M ^-1)	81000¹
Excitation (nm)	569
Emission (nm)	589
Quantum yield	0.15¹

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

1783.14

Absorbance (nm)

573

Correction Factor (260 nm)

0.671

Correction Factor (280 nm)

0.406

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

81000¹

Excitation (nm)

569

Emission (nm)

589

Quantum yield

0.15¹

mFluor™ Blue 590 dye can be well excited with blue laser at 488 nm. It has a huge Stokes shift with emission ~590 nm. mFluor™ Blue 590 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Blue 590 dyes are well excited at 488 nm to give red fluorescence. mFluor™ Blue 590 dye and conjugates are excellent blue laser reagents for flow cytometry detections. Compared to RPE, mFluor™ Blue 590 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 590 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ Blue 590 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 590 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 590 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	56.081 µL	280.404 µL	560.808 µL	2.804 mL	5.608 mL
5 mM	11.216 µL	56.081 µL	112.162 µL	560.808 µL	1.122 mL
10 mM	5.608 µL	28.04 µL	56.081 µL	280.404 µL	560.808 µL

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)	573
Correction Factor (260 nm)	0.671
Correction Factor (280 nm)	0.406
Extinction coefficient (cm ^-1 M ^-1)	81000¹
Excitation (nm)	569
Emission (nm)	589
Quantum yield	0.15¹

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 580 SE	485	580	40000¹	0.363	0.247
mFluor™ Blue 620 SE	589	616	98000¹	0.683	0.849
mFluor™ Violet 590 SE	564	591	90000¹	0.632	0.329

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

Multi-set Pre-processing of Multicolor Flow Cytometry Data.
Authors: Folcarelli, Rita and Tinnevelt, Gerjen H and Hilvering, Bart and Wouters, Kristiaan and van Staveren, Selma and Postma, Geert J and Vrisekoop, Nienke and Buydens, Lutgarde M C and Koenderman, Leo and Jansen, Jeroen J
Journal: Scientific reports (2020): 9716

Immunophenotyping of A20 haploinsufficiency by multicolor flow cytometry.
Authors: Kadowaki, Tomonori and Ohnishi, Hidenori and Kawamoto, Norio and Kadowaki, Saori and Hori, Tomohiro and Nishimura, Kenichi and Kobayashi, Chie and Shigemura, Tomonari and Ogata, Shohei and Inoue, Yuzaburo and Hiejima, Eitaro and Izawa, Kazushi and Matsubayashi, Tadashi and Matsumoto, Kazuaki and Imai, Kohsuke and Nishikomori, Ryuta and Ito, Shuichi and Kanegane, Hirokazu and Fukao, Toshiyuki
Journal: Clinical immunology (Orlando, Fla.) (2020): 108441

Post-induction Measurable Residual Disease Using Multicolor Flow Cytometry Is Strongly Predictive of Inferior Clinical Outcome in the Real-Life Management of Childhood T-Cell Acute Lymphoblastic Leukemia: A Study of 256 Patients.
Authors: Tembhare, Prashant R and Narula, Gaurav and Khanka, Twinkle and Ghogale, Sitaram and Chatterjee, Gaurav and Patkar, Nikhil V and Prasad, Maya and Badrinath, Yajamanam and Deshpande, Nilesh and Gudapati, Pratyusha and Verma, Shefali and Sanyal, Mahima and Kunjachan, Florence and Mangang, Gunit and Gujral, Sumeet and Banavali, Shripad and Subramanian, Papagudi G
Journal: Frontiers in oncology (2020): 577

Analysis of the immune status from peripheral whole blood with a single-tube multicolor flow cytometry assay.
Authors: Donaubauer, Anna-Jasmina and Becker, Ina and Rühle, Paul F and Fietkau, Rainer and Gaipl, Udo S and Frey, Benjamin
Journal: Methods in enzymology (2020): 389-415

Combination of multicolor flow cytometry for circulating lymphoma cells and tests for the RHOAG17V and IDH2R172 hot-spot mutations in plasma cell-free DNA as liquid biopsy for the diagnosis of angioimmunoblastic T-cell lymphoma.
Authors: Hayashida, Masahiko and Maekawa, Fumiyo and Chagi, Yoshinari and Iioka, Futoshi and Kobashi, Yoichiro and Watanabe, Mikio and Ohno, Hitoshi
Journal: Leukemia & lymphoma (2020): 1-10

Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry.
Authors: Galvin, Amy and Weglarz, Meredith and Folz-Donahue, Kat and Handley, Maris and Baum, Misa and Mazzola, Michael and Litwa, Hannah and Scadden, David T and Silberstein, Lev
Journal: Current protocols in cytometry (2019): e50

Immunophenotyping of Tissue Samples Using Multicolor Flow Cytometry.
Authors: Sykora, Martina M and Reschke, Markus
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 253-268

One-Tube Multicolor Flow Cytometry Assay (OTMA) for Comprehensive Immunophenotyping of Peripheral Blood.
Authors: Donaubauer, Anna-Jasmina and Rühle, Paul F and Becker, Ina and Fietkau, Rainer and Gaipl, Udo S and Frey, Benjamin
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 189-212

Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry.
Authors: Cunningham, Rachel A and Holland, Martha and McWilliams, Emily and Hodi, Frank Stephen and Severgnini, Mariano
Journal: Journal of biological methods (2019): e114

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells.
Authors: Wei, Chin-Wen and Zhou, Tyng-An and Dzhagalov, Ivan L and Hsu, Chia-Lin
Journal: Journal of visualized experiments : JoVE (2019)