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mFluor™ UV375 SE

<strong>Top)</strong> Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature. <strong>Bottom)</strong> Flow cytometry analysis of whole blood cells stained with mFluor™ UV375 Anti-human CD4 conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the mFluor™ UV375 specific UV2-A channel.
<strong>Top)</strong> Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature. <strong>Bottom)</strong> Flow cytometry analysis of whole blood cells stained with mFluor™ UV375 Anti-human CD4 conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the mFluor™ UV375 specific UV2-A channel.
Flow cytometry analysis of PBMC stained with mFluor™ UV375 anti-human CD8 *SK1* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the mFluor™ UV375 specific UV1-A channel.
mFluor™ UV375 dyes are well excited by UV laser of 355 nm with emission centered around 375 nm, which perfectly matches the filter set of 379/28 nm used for BUV 395. mFluor™ UV375 NHS esters (or succinimidyl esters) is the most convenient tool for conjugating MFUV375 to a peptide, protein, antibody, amino-modified oligonucleotide or nucleic acid. The 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.
Ordering information
Price ()
Catalog Number1135
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight782.95
SolventDMSO
Spectral properties
Absorbance (nm)342
Correction Factor (260 nm)0.099
Correction Factor (280 nm)0.138
Extinction coefficient (cm -1 M -1)300001
Excitation (nm)351
Emission (nm)387
Quantum yield0.941
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
782.95
Absorbance (nm)
342
Correction Factor (260 nm)
0.099
Correction Factor (280 nm)
0.138
Extinction coefficient (cm -1 M -1)
300001
Excitation (nm)
351
Emission (nm)
387
Quantum yield
0.941
AAT Bioquest's mFluor™ dyes are developed for multicolor flow cytometry-focused applications. These dyes have large Stokes Shifts, and can be well excited by the laser lines of flow cytometers (e.g., 355 nm, 405 nm, 488 nm and 633 nm). mFluor™ UV375 (MFUV375) dyes have fluorescence excitation and emission maxima of ~355 nm and ~375 nm respectively. These spectral characteristics make them an excellent alternative to BD Biosciences' BUV395. mFluor™ UV375 dyes are well excited by UV laser of 355 nm with emission centered around 375 nm, which perfectly matches the filter set of 379/28 nm used for BUV 395. In contrast to polymer-based BUV 395 dye, MFUV375 is a small organic molecule that can be readily conjugated to antibodies. It is significantly dimmer than BUV395, might be used for brighter markers (such as CD4).

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™ UV375 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ UV375 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™ UV375 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™ UV375 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 mM127.722 µL638.61 µL1.277 mL6.386 mL12.772 mL
5 mM25.544 µL127.722 µL255.444 µL1.277 mL2.554 mL
10 mM12.772 µL63.861 µL127.722 µL638.61 µL1.277 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

Absorbance (nm)342
Correction Factor (260 nm)0.099
Correction Factor (280 nm)0.138
Extinction coefficient (cm -1 M -1)300001
Excitation (nm)351
Emission (nm)387
Quantum yield0.941

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
mFluor™ UV460 SE3584561500010.8610.350.134
mFluor™ UV420 SE353421750001---
mFluor™ UV455 SE3574612000010.4210.6510.406
mFluor™ UV520 SE503524800001-0.4950.518
mFluor™ UV540 SE5425609000010.3510.6340.463
mFluor™ UV610 SE5906099000010.250.9490.904

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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