mFluor™ Violet 530 SE
Ordering information
Price | |
Catalog Number | |
Unit Size | |
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
Quotation | Request |
International | See distributors |
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 | ![]() ![]() |
See also: Amine Reactive Dyes and Probes for Conjugation, Bioconjugation, Chemical Reagents, Dyes by Functional Group, Flow Cytometry Reagents, mFluor™ Dyes and Kits
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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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 | 421 | 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 |
Show More (2) |
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
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.
Authors: Soh, Kah Teong and Tario, Joseph D and Hahn, Theresa E and Hillengass, Jens and McCarthy, Philip L and Wallace, Paul K
Journal: Cytometry. Part B, Clinical cytometry (2020): 161-173
Authors: Soh, Kah Teong and Tario, Joseph D and Hahn, Theresa E and Hillengass, Jens and McCarthy, Philip L and Wallace, Paul K
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
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
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
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
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
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].
Authors: Wang, Hui and Liu, Lu and Liu, Fang and Chen, Li-Juan and Qu, Xiao-Yan and Li, Jian-Yong and Wu, Yu-Jie
Journal: Zhongguo shi yan xue ye xue za zhi (2017): 1069-1073
Authors: Wang, Hui and Liu, Lu and Liu, Fang and Chen, Li-Juan and Qu, Xiao-Yan and Li, Jian-Yong and Wu, Yu-Jie
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
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.
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Journal: Thrombosis research (2015): 786-96
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Journal: Thrombosis research (2015): 786-96
Application notes
A New Protein Crosslinking Method for Labeling and Modifying Antibodies
Abbreviation of Common Chemical Compounds Related to Peptides
Bright Tide Fluor™-Based Fluorescent Peptides and Their Applications In Drug Discovery and Disease Diagnosis
FITC (Fluorescein isothiocyanate)
Fluorescein isothiocyanate (FITC)
Abbreviation of Common Chemical Compounds Related to Peptides
Bright Tide Fluor™-Based Fluorescent Peptides and Their Applications In Drug Discovery and Disease Diagnosis
FITC (Fluorescein isothiocyanate)
Fluorescein isothiocyanate (FITC)