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mFluor™ Blue 570 SE

Flow cytometry analysis of whole blood stained with mFluor™ Blue 570 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 570 specific B4-A channel.
Flow cytometry analysis of whole blood stained with mFluor™ Blue 570 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 570 specific B4-A channel.
Flow cytometry analysis of whole blood stained with mFluor™ Blue 570 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Blue 570 specific B4-A channel.
Fluorescent dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating dyes to a peptide, protein, antibody, amino-modified oligonucleotide or nucleic acid. NHS esters react readily with the primary amines (R-NH<sub>2</sub>) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.
Spectral signature of mFluor™ Blue 570 dye. Data acquired on a 4-laser Cytek Aurora and normal human peripheral blood cells stained with clone SK3 (CD4) conjugated to mFluor™ Blue 570 dye (Cat. No. 100420T0) were used for analysis.
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Physical properties
Molecular weight1834.25
SolventDMSO
Spectral properties
Absorbance (nm)553
Correction Factor (260 nm)0.228
Correction Factor (280 nm)0.179
Extinction coefficient (cm -1 M -1)1200001
Excitation (nm)505
Emission (nm)564
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
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Molecular weight
1834.25
Absorbance (nm)
553
Correction Factor (260 nm)
0.228
Correction Factor (280 nm)
0.179
Extinction coefficient (cm -1 M -1)
1200001
Excitation (nm)
505
Emission (nm)
564
mFluor™ Blue 570 dyes are an excellent alternative to RPE since they have the spectral properties equivalent to those of RPE conjugates. mFluor™ Blue 570 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Blue 570 dyes are well excited at 488 nm to give red fluorescence (compatible with TRITC filter). mFluor™ Blue 570 dye and conjugates are excellent blue laser reagents for flow cytometry research. Compared to RPE, mFluor™ Blue 570 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.

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™ Blue 570 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ Blue 570 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 570 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
  1. 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.
  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 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™ Blue 570 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 mM54.518 µL272.591 µL545.182 µL2.726 mL5.452 mL
5 mM10.904 µL54.518 µL109.036 µL545.182 µL1.09 mL
10 mM5.452 µL27.259 µL54.518 µL272.591 µL545.182 µL

Molarity calculator

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Spectrum


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spectrum

Spectral properties

Absorbance (nm)553
Correction Factor (260 nm)0.228
Correction Factor (280 nm)0.179
Extinction coefficient (cm -1 M -1)1200001
Excitation (nm)505
Emission (nm)564

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (260 nm)Correction Factor (280 nm)
mFluor™ Blue 570 acid55356512000010.2280.179
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 583 SE4985854500011.170.35
mFluor™ Blue 615 SE510615400001--

Images


Citations


View all 3 citations: Citation Explorer
Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769
Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018
Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function
Authors: Piccard, Helene and Berghmans, Nele and Korpos, Eva and Dillen, Chris and Aelst, Ilse Van and Li, S and ra , undefined and Martens, Erik and Liekens, S and ra , undefined and Noppen, Sam and Damme, Jo Van and others, undefined
Journal: International Journal of Cancer (2012): E425--E436

References


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