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mFluor™ Green 615 SE

mFluor™ Green 615 dye can be well excited with green laser at 532 nm. It has a huge Stokes shift with emission ~615 nm. mFluor™ Green 615 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Green 615 dyes are well excited at 532 nm to give red fluorescence. mFluor™ Green 615 dye and conjugates are excellent green laser reagents for flow cytometry detections. Compared to the RPE, mFluor™ Green 615 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. It is also a unique fluorochrome for spectral flow cytometry since there are very few existing dyes that have this spectral profile. In addition, mFluor™ Green 615 dyes are small organic dyes that have much smaller size than the macromolecular RPE, thus potentially less affinity interference for the molecules to be labelled.

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 it is lower than 8.0, adjust it to 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. For optimal labeling results, sodium azide or thimerosal can be removed by dialysis or spin column.

    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™ Green 615 SE stock solution (Solution B)
  1. Add anhydrous DMSO into the vial of mFluor™ Green 615 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 a 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™ Green 615 SE. You might need further optimization for your particular proteins.

Each protein requires a distinct dye/protein ratio, which also depends on the properties of the 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, dilute the dye-protein conjugate with staining buffer and aliquot for multiple uses.

    Note: For longer-term storage, the dye-protein conjugate solution must 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 proteins. Proteins with lower DOS usually have weaker fluorescence intensity, but proteins with higher DOS tend to have reduced fluorescence, too. The optimal DOS for most antibodies is between 2 and 10, depending on the properties of the dye and protein. The following steps are used to determine the DOS of mFluor™ Green 615 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 = 616 nm for mFluor™ Green 615 dyes)

For most spectrophotometers, the sample (from the column fractions) must be diluted with de-ionized water so that the OD 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 612 nm is the maximum absorption of mFluor™ Green 615 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  

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
mFluor™ Blue 615 SE510615400001

References

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Correlative super-resolution microscopy with deep UV reactivation.
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Authors: Göppert, Natalie E and Quader, Sabina and Van Guyse, Joachim F R and Weber, Christine and Kataoka, Kazunori and Schubert, Ulrich S
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Authors: Shih, Cheng-Wei and Chang, Chun-Hui
Journal: Brain structure & function (2023)
A model eye for fluorescent characterization of retinal cultures and tissues.
Authors: Ferraro, G and Gigante, Y and Pitea, M and Mautone, L and Ruocco, G and Di Angelantonio, S and Leonetti, M
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Total Flavonoids of Polygala fallax Hemsl Induce Apoptosis of Human Ectopic Endometrial Stromal Cells through PI3K/AKT/Bcl-2 Signaling Pathway.
Authors: Zhong, Chuanmei and Ju, Gongchenhao and Yang, Sufang and Zhao, Xiangpei and Chen, Jixiang and Li, Ning
Journal: Gynecologic and obstetric investigation (2023): 197-213
Page updated on October 4, 2024

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Physical properties

Molecular weight

815.87

Solvent

DMSO

Spectral properties

Extinction coefficient (cm -1 M -1)

400001

Excitation (nm)

530

Emission (nm)

612

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
Top) The Spectral pattern was generated using a 4-laser spectral cytometer. Four spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to create four distinct emission profiles, which, when combined, yielded the overall spectral signature. Bottom) Flow cytometry analysis of whole blood cells stained with mFluor™ Green 615 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Green 615-specific B6-A channel.
Top) The Spectral pattern was generated using a 4-laser spectral cytometer. Four spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to create four distinct emission profiles, which, when combined, yielded the overall spectral signature. Bottom) Flow cytometry analysis of whole blood cells stained with mFluor™ Green 615 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Green 615-specific B6-A channel.
Top) The Spectral pattern was generated using a 4-laser spectral cytometer. Four spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to create four distinct emission profiles, which, when combined, yielded the overall spectral signature. Bottom) Flow cytometry analysis of whole blood cells stained with mFluor™ Green 615 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor™ Green 615-specific B6-A channel.

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