mFluor™ Red 780 Maleimide

mFluor™ Red 780 dyes are an excellent alternative to APC-Alexa Fluor® 750 tandems since they have the spectral properties equivalent to those of APC-Alexa Fluor® 750 conjugates. mFluor™ Red 780 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Red 780 dyes are well excited at 633 nm or 647 nm to give red fluorescence (compatible with Cy7® filter). mFluor™ Red 780 dyes and conjugates are excellent red laser reagents for flow cytometry research. Compared to APC-Alexa Fluor® 750 tandems, mFluor™ Red 780 dyes are much more photostable, making them readily available for fluorescence imaging applications while it is very difficult to use the APC-Alexa Fluor® 750 conjugates for fluorescence imaging applications due to the rapid photobleaching of APC-Alexa Fluor® 750 tandems. mFluor™ Red 780 maleimide is stable and highly reacts with thiol-containing biomolecules such as reduced antibodies.

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. mFluor™ Red 780 maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ Red 780 maleimide 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 upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.

2. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 100 mM MES buffer with pH ~6.0) 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 6.5 ± 0.5.
Note     Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well.
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.
Optional: if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:

Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
Make IgG solution in 20 mM DTT: add 20 µL of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol).
Note     IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column.
Note     The reduction can be carried out in almost any buffers from pH 7-7.5, e.g., MES, phosphate or TRIS buffers.
Note     Steps 3 and 4 can be replaced by dialysis.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with mFluor™ Red 780 maleimide. 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.

Spectrum

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Correction Factor (260 nm)	Correction Factor (280 nm)
mFluor™ Red 780 SE	629	767	90000¹	0.101	0.116
mFluor™ Red 780 Styramide	629	767	90000¹	0.101	0.116

References

View all 30 references: Citation Explorer

Creation of a Nanobody-Based Fluorescent Immunosensor Mini Q-body for Rapid Signal-On Detection of Small Hapten Methotrexate.
Authors: Inoue, Akihito and Ohmuro-Matsuyama, Yuki and Kitaguchi, Tetsuya and Ueda, Hiroshi
Journal: ACS sensors (2020)

Synthesis of Quenchbodies for One-Pot Detection of Stimulant Drug Methamphetamine.
Authors: Jeong, Hee-Jin and Dong, Jinhua and Yeom, Chang-Hun and Ueda, Hiroshi
Journal: Methods and protocols (2020)

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging.
Authors: Alam, Md Kausar and El-Sayed, Ayman and Barreto, Kris and Bernhard, Wendy and Fonge, Humphrey and Geyer, C Ronald
Journal: Molecular imaging and biology (2019): 54-66

Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG-Salt Aqueous Two-Phase Systems for Rapid Screening Applications.
Authors: São Pedro, Mariana N and Azevedo, Ana M and Aires-Barros, Maria R and Soares, Ruben R G
Journal: Biotechnology journal (2019): e1800640

Optimizing leading edge F-actin labeling using multiple actin probes, fixation methods and imaging modalities.
Authors: DesMarais, Vera and Eddy, Robert J and Sharma, Ved P and Stone, Orrin and Condeelis, John S
Journal: BioTechniques (2019): 113-119

Page updated on October 22, 2024

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Physical properties

Molecular weight	1168.28
Solvent	DMSO

Spectral properties

Absorbance (nm)	630
Correction Factor (260 nm)	0.101
Correction Factor (280 nm)	0.116
Extinction coefficient (cm ^-1 M ^-1)	90000¹
Excitation (nm)	629
Emission (nm)	767

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501