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mFluor™ Violet 550 SE
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., 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. mFluor™ Violet 550 dyes have fluorescence excitation and emission maxima of ~405 nm and ~550 nm respectively. These spectral characteristics make them a unique color for flow cytometry application. mFluor™ Violet 550 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. mFluor™ Violet 550 SE provides a convenient tool to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) for flow cytometric applications with the violet 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.
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.
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.
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 550 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ Violet 550 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 550 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.
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.
Spectrum
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 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 SE | 505 | 525 | 200001 | - | - | - |
Show More (1) | ||||||
References
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Authors: Tsai, Wanxia Li and Vian, Laura and Giudice, Valentina and Kieltyka, Jacqueline and Liu, Christine and Fonseca, Victoria and Gazaniga, Nathalia and Gao, Shouguo and Kajigaya, Sachiko and Young, Neal S and Biancotto, Angélique and Gadina, Massimo
Journal: Journal of immunological methods (2020): 112667
Authors: Tsai, Wanxia Li and Vian, Laura and Giudice, Valentina and Kieltyka, Jacqueline and Liu, Christine and Fonseca, Victoria and Gazaniga, Nathalia and Gao, Shouguo and Kajigaya, Sachiko and Young, Neal S and Biancotto, Angélique and Gadina, Massimo
Journal: Journal of immunological methods (2020): 112667
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
First record of Doleschallia tongana (Lepidoptera: Nymphalidae) for Guam Island.
Authors: Manuel, Jake and Tennent, W John and Buden, Donald W and Moore, Aubrey
Journal: F1000Research (2018): 366
Authors: Manuel, Jake and Tennent, W John and Buden, Donald W and Moore, Aubrey
Journal: F1000Research (2018): 366
Optimization and standardization of fluorescent cell barcoding for multiplexed flow cytometric phenotyping.
Authors: Giudice, Valentina and Feng, Xingmin and Kajigaya, Sachiko and Young, Neal S and Biancotto, Angélique
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2017): 694-703
Authors: Giudice, Valentina and Feng, Xingmin and Kajigaya, Sachiko and Young, Neal S and Biancotto, Angélique
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2017): 694-703
Quantification of mitochondrial reactive oxygen species in living cells by using multi-laser polychromatic flow cytometry.
Authors: De Biasi, Sara and Gibellini, Lara and Bianchini, Elena and Nasi, Milena and Pinti, Marcello and Salvioli, Stefano and Cossarizza, Andrea
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2016): 1106-1110
Authors: De Biasi, Sara and Gibellini, Lara and Bianchini, Elena and Nasi, Milena and Pinti, Marcello and Salvioli, Stefano and Cossarizza, Andrea
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2016): 1106-1110
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