mFluor™ Blue 630 SE

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	709.86
Solvent	DMSO

Spectral properties

Absorbance (nm)	472
Correction Factor (260 nm)	0.197
Correction Factor (280 nm)	0.275
Extinction coefficient (cm ^-1 M ^-1)	49000¹
Excitation (nm)	470
Emission (nm)	632

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

Alternative formats

Overview SDS Protocol

Molecular weight

709.86

Absorbance (nm)

472

Correction Factor (260 nm)

0.197

Correction Factor (280 nm)

0.275

Extinction coefficient (cm ^-1 M ^-1)

49000¹

Excitation (nm)

470

Emission (nm)

632

mFluor™ Blue 630 dye can be well excited with blue laser at 488 nm. It has a huge Stokes shift with emission ~630 nm. mFluor™ Blue 630 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Blue 630 dyes are well excited at 488 nm to give red fluorescence. mFluor™ Blue 630 dye and conjugates are excellent blue laser reagents for flow cytometry detections. Compared to RPE, mFluor™ Blue 630 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.

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 630 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ Blue 630 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 630 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

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.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of mFluor™ Blue 630 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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	140.873 µL	704.364 µL	1.409 mL	7.044 mL	14.087 mL
5 mM	28.175 µL	140.873 µL	281.746 µL	1.409 mL	2.817 mL
10 mM	14.087 µL	70.436 µL	140.873 µL	704.364 µL	1.409 mL

Molarity calculator

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Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Absorbance (nm)	472
Correction Factor (260 nm)	0.197
Correction Factor (280 nm)	0.275
Extinction coefficient (cm ^-1 M ^-1)	49000¹
Excitation (nm)	470
Emission (nm)	632

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
mFluor™ Green 630 SE	537	657	51000¹
mFluor™ Yellow 630 SE	570	632	110000¹

Images

Figure 1. 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-NH₂) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.

References

View all 19 references: Citation Explorer

A Comprehensive Workflow for Applying Single-Cell Clustering and Pseudotime Analysis to Flow Cytometry Data.
Authors: Melsen, Janine E and van Ostaijen-Ten Dam, Monique M and Lankester, Arjan C and Schilham, Marco W and van den Akker, Erik B
Journal: Journal of immunology (Baltimore, Md. : 1950) (2020)

High-Dimensional Data Analysis Algorithms Yield Comparable Results for Mass Cytometry and Spectral Flow Cytometry Data.
Authors: Ferrer-Font, Laura and Mayer, Johannes U and Old, Samuel and Hermans, Ian F and Irish, Jonathan and Price, Kylie M
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

HTLV infected individuals have increased B-cell activation and proinflammatory regulatory T-cells.
Authors: Kjerulff, Bertram and Petersen, Mikkel Steen and Rodrigues, Candida Medina and da Silva Té, David and Christiansen, Mette and Erikstrup, Christian and Hønge, Bo Langhoff
Journal: Immunobiology (2020): 151878

Multispectral Flow Cytometry: Unaddressed Issues and Recommendations for Improvement.
Authors: Parks, David R
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

High-dimensional analysis of intestinal immune cells during helminth infection.
Authors: Ferrer-Font, Laura and Mehta, Palak and Harmos, Phoebe and Schmidt, Alfonso J and Chappell, Sally and Price, Kylie M and Hermans, Ian F and Ronchese, Franca and le Gros, Graham and Mayer, Johannes U
Journal: eLife (2020)

Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry.
Authors: Ferrer-Font, Laura and Pellefigues, Christophe and Mayer, Johannes U and Small, Sam J and Jaimes, Maria C and Price, Kylie M
Journal: Current protocols in cytometry (2020): e70

Phenotypic Analysis of the Mouse Hematopoietic Hierarchy Using Spectral Cytometry: From Stem Cell Subsets to Early Progenitor Compartments.
Authors: Solomon, Michael and DeLay, Monica and Reynaud, Damien
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

Acquisition of High-Quality Spectral Flow Cytometry Data.
Authors: Fox, Amy and Dutt, Taru S and Karger, Burton and Obregón-Henao, Andrés and Anderson, G Brooke and Henao-Tamayo, Marcela
Journal: Current protocols in cytometry (2020): e74

Spectral flow cytometry-Quo vadimus?
Authors: Robinson, J Paul
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2019): 823-824

Time-Delayed Integration-Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping.
Authors: Hu, Wenting and Soper, Steven A and Jackson, J Matt
Journal: Analytical chemistry (2019): 4656-4664