logo
AAT Bioquest

mFluor™ Blue 585 SE

Flow cytometry analysis of whole blood cells stained with mFluor™ Blue 585 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 585 specific B4-A channel.
Flow cytometry analysis of whole blood cells stained with mFluor™ Blue 585 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 585 specific B4-A channel.
Flow cytometry analysis of whole blood cells stained with mFluor™ Blue 585 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 585 specific B4-A channel.
Ordering information
Price
Catalog Number
Unit Size
Quantity
Add to cart
Additional ordering information
Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Physical properties
Molecular weight835.92
SolventDMSO
Spectral properties
Extinction coefficient (cm -1 M -1)450001
Excitation (nm)491
Emission (nm)578
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
Related products
mFluor™ Violet 450 SE
mFluor™ Violet 510 SE
mFluor™ Violet 540 SE
mFluor™ Green 620 SE
mFluor™ Yellow 630 SE
mFluor™ Red 700 SE
mFluor™ Red 780 SE
mFluor™ Red 780 amine
mFluor™ Violet 450-VAD-FMK
mFluor™ 510-VAD-FMK
mFluor™ Violet 450-streptavidin conjugate
mFluor™ Violet 510-streptavidin conjugate
mFluor™ Violet 540-streptavidin conjugate
mFluor™ Blue 570-streptavidin conjugate
mFluor™ Green 620-streptavidin conjugate
mFluor™ Yellow 630-streptavidin conjugate
mFluor™ Red 700-streptavidin conjugate
mFluor™ Red 780-streptavidin conjugate
mFluor™ Violet 450-dUTP *1 mM in Tris Buffer (pH 7.5)*
mFluor™ Violet 450 acid
mFluor™ Violet 510 acid
mFluor™ Violet 540 acid
mFluor™ Blue 570 acid
mFluor™ Green 620 acid
mFluor™ Yellow 630 acid
mFluor™ Red 700 acid
mFluor™ Violet 500 SE
mFluor™ UV375 SE
mFluor™ Red 780 acid
mFluor™ UV460 SE
mFluor™ Violet 610 SE
mFluor™ Green 630 SE
mFluor™ Red 780 Maleimide
mFluor™ Violet 550 SE
mFluor™ Violet 505 SE
mFluor™ Violet 590 SE
mFluor™ Violet 545 SE
mFluor™ UV420 SE
mFluor™ UV455 SE
mFluor™ UV520 SE
mFluor™ UV540 SE
mFluor™ UV610 SE
mFluor™ Violet 450 Azide
mFluor™ Violet 450 maleimide
mFluor™ UV 375 Biotin Conjugate
mFluor™ UV 460 Biotin Conjugate
mFluor™ Violet 500 Biotin Conjugate
mFluor™ Violet 540 Biotin Conjugate
mFluor™ Red 780 Biotin Conjugate
mFluor™ Violet 530 SE
mFluor™ Violet 530 maleimide
mFluor™ Violet 480 SE
mFluor™ Blue 660 tyramide
mFluor™ Violet 450-PEG4-Biotin Conjugate
mFluor™ Violet 450-Wheat Germ Agglutinin (WGA) Conjugate
mFluor™ Violet 500-Wheat Germ Agglutinin (WGA) Conjugate
mFluor™ Violet 540-Wheat Germ Agglutinin (WGA) Conjugate
mFluor™ Blue 580 Styramide
mFluor™ Blue 630 Styramide
mFluor™ Blue 660 Styramide
mFluor™ Green 620 Styramide
mFluor™ Red 780 Styramide
mFluor™ Violet 540 Styramide
mFluor™ Violet 545 Styramide
mFluor™ Violet 610 Styramide
mFluor™ UV455-streptavidin conjugate
mFluor™ Violet 545-streptavidin conjugate
mFluor™ Violet 550-streptavidin conjugate
mFluor™ Violet 590-streptavidin conjugate
mFluor™ Violet 610-streptavidin conjugate
mFluor™ UV 375 goat anti-mouse IgG (H+L)
mFluor™ UV 375 goat anti-rabbit IgG (H+L)
mFluor™ UV 375 goat anti-rabbit IgG (H+L) *Cross-Absorbed*
mFluor™ UV 375 goat anti-mouse IgG (H+L) *Cross-Absorbed*
mFluor™ Violet 510 goat anti-mouse IgG (H+L)
mFluor™ Violet 510 goat anti-mouse IgG (H+L) *Cross-Absorbed*
Show More (66)

OverviewpdfSDSpdfProtocol


Molecular weight
835.92
Extinction coefficient (cm -1 M -1)
450001
Excitation (nm)
491
Emission (nm)
578
mFluor™ Blue 585 dyes are an excellent alternative to RPE since they have the spectral properties equivalent to those of RPE conjugates. mFluor™ Blue 585 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Blue 585 dyes are well excited at 488 nm to give red fluorescence (compatible with TRITC filter). mFluor™ Blue 585 dye and conjugates are excellent blue laser reagents for flow cytometry applications. Compared to RPE, mFluor™ Blue 585 dyes are much more photostable, making them readily available for fluorescence imaging applications while it is very difficult to use RPE conjugates for fluorescence imaging applications due to the rapid photobleaching of RPE conjugates. mFluor™ Blue 585 are small organic dyes, thus they are much more readily conjugated to proteins with much higher yield than PE. AAT Bioquest's mFluor™ dye family provides the largest collection of fluorescent labeling dyes with large Stokes Shift. They are optimized for labeling proteins, in particular, antibodies. These dyes are bright, photostable and have minimal quenching on proteins. They have been validated for multicolor applications, in particular, for spectral flow cytometric applications. They can be well excited by the major laser lines of spectral flow cytometers (e.g., 350, 405, 488, 555, 633 and 647 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

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.

mFluor™ Blue 585 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ Blue 585 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™ Blue 585 SE. You might need further optimization for your particular proteins.

Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity while the protein conjugates of low dye/protein ratio give reduced sensitivity.

Run conjugation reaction
  1. Use a 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 using a 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too low or too high, determine the optimal dye/protein ratio at 5:1, 15:1, and 20:1 respectively.

  2. 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.

  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. 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 needs to be diluted with staining buffer, and aliquoted for multiple uses.

    Note: For longer-term storage, the dye-protein conjugate solution needs to be concentrated or freeze-dried.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of mFluor™ Blue 585 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 mg0.5 mg1 mg5 mg10 mg
1 mM119.629 µL598.143 µL1.196 mL5.981 mL11.963 mL
5 mM23.926 µL119.629 µL239.257 µL1.196 mL2.393 mL
10 mM11.963 µL59.814 µL119.629 µL598.143 µL1.196 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)450001
Excitation (nm)491
Emission (nm)578

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (260 nm)Correction Factor (280 nm)
mFluor™ Blue 570 SE55356512000010.2280.179
mFluor™ Blue 630 SE4706324900010.1970.275
mFluor™ Blue 660 SE4816632600010.3380.32
mFluor™ Blue 580 SE4855804000010.3630.247
mFluor™ Blue 590 SE5695898100010.6710.406
mFluor™ Blue 620 SE5896169800010.6830.849
mFluor™ Blue 583 SE4985854500011.170.35

Images


References


View all 4 references: Citation Explorer
Associations between Hypertriglyceridemia and Circulating Neutrophil Subpopulation in Patients with Dyslipidemia.
Authors: Genkel, Vadim and Dolgushin, Ilya and Baturina, Irina and Savochkina, Albina and Kuznetsova, Alla and Pykhova, Lubov and Shaposhnik, Igor
Journal: International journal of inflammation (2021): 6695468
Fluorochrome choices for multi-color flow cytometry.
Authors: Flores-Montero, Juan and Kalina, Tomas and Corral-Mateos, Alba and Sanoja-Flores, Luzalba and Pérez-Andrés, Martin and Martin-Ayuso, Marta and Sedek, Lukasz and Rejlova, Katerina and Mayado, Andrea and Fernández, Paula and van der Velden, Vincent and Bottcher, Sebastian and van Dongen, Jaques J M and Orfao, Alberto
Journal: Journal of immunological methods (2019): 112618
Unexpected interference in cell surface staining by monoclonal antibodies to unrelated antigens.
Authors: De Vita, Martina and Catzola, Valentina and Buzzonetti, Alexia and Fossati, Marco and Battaglia, Alessandra and Zamai, Loris and Fattorossi, Andrea
Journal: Cytometry. Part B, Clinical cytometry (2015): 352-4
Unexpected interference in cell surface staining by monoclonal antibodies to unrelated antigens.
Authors: De Vita, Martina and Catzola, Valentina and Buzzonetti, Alexia and Fossati, Marco and Battaglia, Alessandra and Zamai, Loris and Fattorossi, Andrea
Journal: Cytometry. Part B, Clinical cytometry (2014)