mFluor™ UV460 SE

AAT Bioquest\'s 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., 355 nm, 405 nm, 488 nm and 633 nm). mFluor™ UV460 (MFUV460) dyes are well excited by UV excitation with emission at ~460 nm. MFUV460 has the spectral properties almost identical to those of Marina Blue® dye. These spectral characteristics make them an excellent alternative to Marina Blue® dye. Marina Blue® is the trademark of ThermoFisher.

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™ UV460 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ UV460 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™ UV460 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™ UV460 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	272.287 µL	1.361 mL	2.723 mL	13.614 mL	27.229 mL
5 mM	54.457 µL	272.287 µL	544.573 µL	2.723 mL	5.446 mL
10 mM	27.229 µL	136.143 µL	272.287 µL	1.361 mL	2.723 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

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™ UV375 SE	351	387	30000¹	0.94¹	0.099	0.138
mFluor™ UV420 SE	353	421	75000¹	-	-	-
mFluor™ UV455 SE	357	461	20000¹	0.42¹	0.651	0.406
mFluor™ UV520 SE	503	524	80000¹	-	0.495	0.518
mFluor™ UV540 SE	542	560	90000¹	0.35¹	0.634	0.463
mFluor™ UV610 SE	590	609	90000¹	0.25	0.949	0.904
mFluor™ UV460 maleimide	358	456	15000¹	0.86¹	0.35	0.134

Citations

View all 11 citations: Citation Explorer

Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes
Authors: Cabre, E. J., Martinez-Calle, M., Prieto, M., Fedorov, A., Olmeda, B., Loura, L. M. S., Perez-Gil, J.
Journal: J Biol Chem (2018): 9399-9411

Molecular characterization of DXCF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor
Authors: Hasegawa, M., Fushimi, K., Miyake, K., Nakajima, T., Oikawa, Y., Enomoto, G., Sato, M., Ikeuchi, M., Narikawa, R.
Journal: J Biol Chem (2018): 1713-1727

Low blue carbon storage in eelgrass (Zostera marina) meadows on the Pacific Coast of Canada
Authors: Postlethwaite, V. R., McGowan, A. E., Kohfeld, K. E., Robinson, C. L. K., Pellatt, M. G.
Journal: PLoS One (2018): e0198348

Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion
Authors: Liu, X., Seven, A. B., Xu, J., Esser, V., Su, L., Ma, C., Rizo, J.
Journal: Nat Protoc (2017): 2014-2028

A biliverdin-binding cyanobacteriochrome from the chlorophyll d-bearing cyanobacterium Acaryochloris marina
Authors: Narikawa, R., Nakajima, T., Aono, Y., Fushimi, K., Enomoto, G., Ni Ni, Win, Itoh, S., Sato, M., Ikeuchi, M.
Journal: Sci Rep (2015): 7950

Page updated on November 21, 2024

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Catalog Number	1136
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Physical properties

Molecular weight	367.26
Solvent	DMSO

Spectral properties

Absorbance (nm)	364
Correction Factor (260 nm)	0.35
Correction Factor (280 nm)	0.134
Extinction coefficient (cm ^-1 M ^-1)	15000¹
Excitation (nm)	358
Emission (nm)	456
Quantum yield	0.86¹