mFluor™ Violet 530 SE

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

Physical properties

Molecular weight	1174.17
Solvent	DMSO

Spectral properties

Absorbance (nm)	398
Extinction coefficient (cm ^-1 M ^-1)	20000¹
Excitation (nm)	393
Emission (nm)	543

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

Overview SDS Protocol

Molecular weight

1174.17

Absorbance (nm)

398

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

20000¹

Excitation (nm)

393

Emission (nm)

543

mFluor™ Violet 530 dyes have fluorescence excitation and emission maxima of ~405 nm and ~530 nm respectively. These spectral characteristics make them a unique color for flow cytometry application. mFluor™ Violet 530 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. It provides a convenient tool to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) for flow cytometric applications with the violet laser excitation, in particular suitable for spectral flow cytometric applications. 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™ Violet dyes are optimized to be excited with a Violet laser at 405 nm. AAT Bioquest offers the largest collection of fluorescent dyes that are excited by Violet laser at 405 nm.

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™ Violet 530 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ Violet 530 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™ Violet 530 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™ Violet 530 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	85.167 µL	425.833 µL	851.665 µL	4.258 mL	8.517 mL
5 mM	17.033 µL	85.167 µL	170.333 µL	851.665 µL	1.703 mL
10 mM	8.517 µL	42.583 µL	85.167 µL	425.833 µL	851.665 µL

Molarity calculator

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

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Spectral properties

Absorbance (nm)	398
Extinction coefficient (cm ^-1 M ^-1)	20000¹
Excitation (nm)	393
Emission (nm)	543

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
mFluor™ Violet 450 SE	406	445	35000¹	0.81¹	0.338	0.078
mFluor™ Violet 510 SE	412	505	25000¹	0.86¹	0.464	0.366
mFluor™ Violet 540 SE	402	535	18000¹	0.21¹	1.326	0.543
mFluor™ Violet 500 SE	410	501	25000¹	0.81¹	0.769	0.365
mFluor™ Violet 610 SE	594	612	90000¹	0.3¹	0.532	0.66
mFluor™ Violet 550 SE	527	550	90000¹	0.31¹	0.474	0.306
mFluor™ Violet 505 SE	393	504	40000¹	0.45¹	0.888	0.403
mFluor™ Violet 590 SE	564	591	90000¹	0.22¹	0.632	0.329
mFluor™ Violet 545 SE	393	543	20000¹	0.15¹	1.08	0.496
mFluor™ Violet 530 maleimide	393	543	20000¹	-	-	-
mFluor™ Violet 480 SE	404	475	40,000	-	1.23	0.6570
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References

View all 10 references: Citation Explorer

Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG, IgM and IgA using beads covalently coupled to the nucleocapsid protein.
Authors: Zattoni, I F and Huergo, L F and Gerhardt, E C M and Nardin, J M and Dos Santos, A M F and Rego, F G M and Picheth, G and Moure, V R and Valdameri, G
Journal: Letters in applied microbiology (2022): 863-872

Methodological considerations for the high sensitivity detection of multiple myeloma measurable residual disease.
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Journal: Cytometry. Part B, Clinical cytometry (2020): 161-173

Chronic and oxidative stress association with total count of endothelial microvesicles in healthy young male plasma.
Authors: Žėkas, Vytautas and Matuzevičienė, Reda and Karčiauskaitė, Dovilė and Mažeikienė, Asta and Burokienė, Neringa and Radzevičius, Mantas and Janilionienė, Aušra and Linkevičiūtė, Aušra and Kučinskienė, Zita Aušrelė
Journal: Advances in clinical and experimental medicine : official organ Wroclaw Medical University (2019): 683-692

Diet-induced microbial autofluorescence confounds flow cytometry of ex vivo isolated fecal microbes.
Authors: Denu, Lidiya and Lubin, Jean-Bernard and Douglas, Bonnie and Tuluc, Florin and Silverman, Michael A
Journal: European journal of immunology (2019): 2252-2254

Lot-to-lot stability of antibody reagents for flow cytometry.
Authors: Böttcher, Sebastian and van der Velden, Vincent H J and Villamor, Neus and Ritgen, Matthias and Flores-Montero, Juan and Escobar, Hugo Murua and Kalina, Tomas and Brüggemann, Monika and Grigore, Georgiana and Martin-Ayuso, Marta and Lecrevisse, Quentin and Pedreira, Carlos E and van Dongen, Jacques J M and Orfao, Alberto
Journal: Journal of immunological methods (2019): 112294

Improved panels for clinical immune phenotyping: Utilization of the violet laser.
Authors: Ryherd, Mark and Plassmeyer, Matthew and Alexander, Connor and Eugenio, Ines and Kleschenko, Yuliya and Badger, Ariel and Gupta, Raavi and Alpan, Oral and Sønder, Søren Ulrik
Journal: Cytometry. Part B, Clinical cytometry (2018): 671-679

One tube with eight antibodies for 14-part bone marrow leukocyte differential using flow cytometry.
Authors: Jacob, Marie-Christine and Souvignet, Alice and Pont, Julie and Solly, Françoise and Mondet, Julie and Kesr, Sanae and Pernollet, Martine and Dumestre-Perard, Chantal and Campos, Lydia and Cesbron, Jean-Yves
Journal: Cytometry. Part B, Clinical cytometry (2017): 299-309

[Diagnostic Value of CD27 Antigen in Patients with Multiple Myeloma].
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Journal: Zhongguo shi yan xue ye xue za zhi (2017): 1069-1073

Measurement of monocyte-platelet aggregates by imaging flow cytometry.
Authors: Hui, Henry and Fuller, Kathryn and Erber, Wendy N and Linden, Matthew D
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2015): 273-8

Using six-colour flow cytometry to analyse the activation and interaction of platelets and leukocytes--A new assay suitable for bench and bedside conditions.
Authors: Granja, Tiago and Schad, Jessica and Schüssel, Patricia and Fischer, Claudius and Häberle, Helene and Rosenberger, Peter and Straub, Andreas
Journal: Thrombosis research (2015): 786-96