mFluor™ Violet 530 maleimide

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 Maleimide is reasonably stable and shows good reactivity and selectivity with thiol group. It provides a convenient tool to label the reduced form of monoclonal, polyclonal antibodies or other proteins that contains a thiol group. These conjugates are useful 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. mFluor™ Violet 530 maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ Violet 530 maleimide 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 upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.

2. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 100 mM MES buffer with pH ~6.0) 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 6.5 ± 0.5. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well. 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.
Optional: if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:

Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
Make IgG solution in 20 mM DTT: add 20 µL of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol). Note: IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column. Note: The reduction can be carried out in almost any buffers from pH 7-7.5, e.g., MES, phosphate or TRIS buffers. Note: Steps 3 and 4 can be replaced by dialysis.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with mFluor™ Violet 530 maleimide. 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.

Spectrum

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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 maleimide	406	445	35000¹	0.81¹	0.338	0.078
mFluor™ Violet 530 SE	393	543	20000¹	-	-	-
mFluor™ Violet 610 maleimide	594	612	90000¹	0.3¹	0.532	0.66
mFluor™ Violet 500 maleimide	410	501	25000¹	0.81¹	0.769	0.365

References

View all 48 references: Citation Explorer

Automated fluorescence gating and size determination reduce variation in measured concentration of extracellular vesicles by flow cytometry.
Authors: Gankema, A A F and Li, B and Nieuwland, R and van der Pol, E
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2022)

Azo dyes decolorization under high alkalinity and salinity conditions by Halomonas sp. in batch and packed bed reactor.
Authors: Montañez-Barragán, B and Sanz-Martín, J L and Gutiérrez-Macías, P and Morato-Cerro, A and Rodríguez-Vázquez, R and Barragán-Huerta, B E
Journal: Extremophiles : life under extreme conditions (2020): 239-247

Adsorption of Remazol Brilliant Violet-5R Textile Dye from Aqueous Solutions by Using Eggshell Waste Biosorbent.
Authors: Rápó, Eszter and Aradi, László Előd and Szabó, Ábel and Posta, Katalin and Szép, Robert and Tonk, Szende
Journal: Scientific reports (2020): 8385

Process optimization and filtration performance of an anaerobic dynamic membrane bioreactor treating textile wastewaters.
Authors: Yurtsever, Adem and Basaran, Erkan and Ucar, Deniz
Journal: Journal of environmental management (2020): 111114

Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy.
Authors: Schmidt, Alfonso J and Mayer, Johannes U and Wallace, Paul K and Ronchese, Franca and Price, Kylie M
Journal: Current protocols in cytometry (2019): e64

Page updated on November 20, 2024

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