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AAT Bioquest

mFluor™ Blue 583 SE

Flow cytometry analysis of PBMCs stained with mFluor™ Blue 583 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 583 specific B4-A channel.
Flow cytometry analysis of PBMCs stained with mFluor™ Blue 583 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 583 specific B4-A channel.
Flow cytometry analysis of PBMCs stained with mFluor™ Blue 583 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 583 specific B4-A channel.
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Physical properties
Molecular weight770.84
SolventDMSO
Spectral properties
Correction Factor (260 nm)1.17
Correction Factor (280 nm)0.35
Extinction coefficient (cm -1 M -1)450001
Excitation (nm)498
Emission (nm)585
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
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OverviewpdfSDSpdfProtocol


Molecular weight
770.84
Correction Factor (260 nm)
1.17
Correction Factor (280 nm)
0.35
Extinction coefficient (cm -1 M -1)
450001
Excitation (nm)
498
Emission (nm)
585
AAT Bioquest's mFluor™ dyes are developed for multicolor flow cytometry-focused applications, in particular, for spectral fluorescence flow cytometry. mFluor™ Blue 583 dye is well excited with blue laser at 488 nm. It has a huge Stokes shift with emission ~583 nm. mFluor™ Blue 583 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Blue 583 dyes are well excited at 488 nm to give bright red fluorescence. mFluor™ Blue 583 dye and conjugates are excellent blue laser-excitable reagents for flow cytometry applications. It is a unique fluorochrome for spectral flow cytometry since there are very few existing dyes that have this spectral profile. Advances in spectral flow cytometers have expanded applications and capabilities beyond conventional flow cytometry. The new color of mFluor™ Blue 583 provides a new option for spectral flow cytometry analysis, enabling researchers and scientists to investigate an increasing number of molecules of interest. Compared to RPE, mFluor™ Blue 583 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.

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)
  1. 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 583 SE stock solution (Solution B)
  1. Add anhydrous DMSO into the vial of mFluor™ Blue 583 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 the freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol is specifically optimized for the conjugation of goat anti-mouse IgG with mFluor™ Blue 583 SE. Further optimization may be necessary for other 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.

Characterize the Desired Dye-Protein Conjugate

The Degree of Substitution (DOS) is the most important factor for characterizing dye-labeled protein. Proteins of lower DOS usually have weaker fluorescence intensity, but proteins of higher DOS (e.g., DOS > 6) tend to have reduced fluorescence too. The optimal DOS for most antibodies is recommended between 2 and 10, depending on the properties of dye and protein. For effective labeling, the degree of substitution should be controlled to have 6-8 moles of mFluor™ Blue 583 SE to one mole of antibody. The following steps are used to determine the DOS of mFluor™ Blue 583 SE labeled proteins.

Measure absorption

To measure the absorption spectrum of a dye-protein conjugate, it is recommended to keep the sample concentration in the range of 1-10 µM depending on the extinction coefficient of the dye.

Read OD (absorbance) at 280 nm and dye maximum absorption (ƛmax = 583 nm for mFluor™ Blue 583 dye)

For most spectrophotometers, the sample (from the column fractions) needs to be diluted with de-ionized water so that the O.D. values are in the range of 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein, while 583 nm is the maximum absorption of mFluor™ Blue 583 SE. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye. 

Calculate DOS

You can calculate DOS using our tool by following this link: https://www.aatbio.com/tools/degree-of-labeling-calculator

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of mFluor™ Blue 583 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 mM129.729 µL648.643 µL1.297 mL6.486 mL12.973 mL
5 mM25.946 µL129.729 µL259.457 µL1.297 mL2.595 mL
10 mM12.973 µL64.864 µL129.729 µL648.643 µL1.297 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


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spectrum

Spectral properties

Correction Factor (260 nm)1.17
Correction Factor (280 nm)0.35
Extinction coefficient (cm -1 M -1)450001
Excitation (nm)498
Emission (nm)585

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 585 SE491578450001--
mFluor™ Blue 615 SE510615---

Images


References


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