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trFluor™ Eu maleimide

Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.
Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.
Ordering information
Price ()
Catalog Number1434
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 weight1116.96
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.911
Correction Factor (280 nm)0.777
Excitation (nm)346
Emission (nm)617
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

OverviewpdfSDSpdfProtocol


Molecular weight
1116.96
Correction Factor (260 nm)
0.911
Correction Factor (280 nm)
0.777
Excitation (nm)
346
Emission (nm)
617
Many biological compounds present in cells, serum or other biological fluids are naturally fluorescent, and thus the use of conventional, prompt fluorophores leads to serious limitations in assay sensitivity due to the high background caused by the autofluorescence of the biological molecules to be assayed. The use of long-lived fluorophores combined with time-resolved detection (a delay between excitation and emission detection) minimizes prompt fluorescence interferences. Our trFluor™ Eu probes enable time-resolved fluorometry (TRF) for the assays that require high sensitivity. These trFluor™ Eu probes have large Stokes shifts and extremely long emission half-lives when compared to more traditional fluorophores such as Alexa Fluor or cyanine dyes. Compared to the other TRF compounds, our trFluor™ Eu probes have relatively high stability, high emission yield and ability to be linked to biomolecules. Moreover, our trFluor™ Eu probes are insensitive to fluorescence quenching when conjugated to biological polymers such as antibodies.

Platform


Fluorescence microplate reader

Excitation346 nm
Emission617 nm
Cutoff370 nm
Recommended plateSolid black

Example protocol


PREPARATION OF WORKING SOLUTION

Dye labelling solution (7mM)
Add 10 µL DMSO to the vial to make 7 mM dye labeling solution. Note: We recommend preparing fresh dye labelling solution.

SAMPLE EXPERIMENTAL PROTOCOL

Protocol for Labeling Proteins with trFluor™ Eu Maleimide:
  1. Dissolve your thiol-containing protein at concentration 1-10 mg/mL (3-10 mg is the optimal labeling concentration) using PBS buffer (20 mM, pH 7.2).
  2. Mix the trFluor™ Maleimide and protein solution at 20:1 molar ratio of dye/protein, and shake the reaction mixture at room temperature for 2-4 hours in the dark.
  3. Filter the reaction mixture through a protein spin column for 100 µg to 1 mg protein labeling reaction; or purify the conjugate using gel filtration on a properly sized Sephadex G-25 column if the reaction scale is larger than 1 mg.
  4. Collect the desired fractions for your immediate use or freeze dry them for your future use. Note: The trFluor™ conjugate need be used near neutral pH range (6.5 to 7.5). Either acidic or basic pH would reduce its fluorescence intensity. 

Protocol for Labeling Small Molecules with trFluor™ Eu Maleimide:
  1. Dissolve trFluor™ Maleimide (10 -15 mg/mL) and your thiol-contain molecule in DMSO at 1:1.2 molar ratio of dye/ thiol-contain molecule.
  2. Stir the reaction mixture at room temperature for 2-4 hours in the dark.
  3. Purify the conjugate using HPLC (ammonium acetate/water and acetonitrile, pH 7.0).
  4. Collect and pool the desired fractions.
  5. Combine and freeze-dry the pooled fractions. Note: The trFluor™ conjugate need be used near neutral pH range (6.5 to 7.5). Either acidic or basic pH would reduce its fluorescence intensity. Note: These protocols can be used as sample protovols. We recommend to modify as per needed. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of trFluor™ Eu maleimide 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 mM89.529 µL447.644 µL895.287 µL4.476 mL8.953 mL
5 mM17.906 µL89.529 µL179.057 µL895.287 µL1.791 mL
10 mM8.953 µL44.764 µL89.529 µL447.644 µL895.287 µL

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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.911
Correction Factor (280 nm)0.777
Excitation (nm)346
Emission (nm)617

Product family


NameExcitation (nm)Emission (nm)Correction Factor (260 nm)Correction Factor (280 nm)
trFluor™ Tb maleimide3335440.9420.797
trFluor™ Eu DBCO3466170.9110.777

Citations


View all 1 citations: Citation Explorer

References


View all 61 references: Citation Explorer
Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors
Authors: Lo MC, Ngo R, Dai K, Li C, Liang L, Lee J, Emkey R, Eksterowicz J, Ventura M, Young SW, Xiao SH.
Journal: Anal Biochem (2012): 368
Time-Resolved Fluorescence Resonance Energy Transfer as a Versatile Tool in the Development of Homogeneous Cellular Kinase Assays
Authors: Saville L, Spais C, Mason JL, Albom MS, Murthy S, Meyer SL, Ator MA, Angeles TS, Husten J.
Journal: Assay Drug Dev Technol. (2012)
Oligomerization of the serotonin(1A) receptor in live cells: a time-resolved fluorescence anisotropy approach
Authors: Paila YD, Kombrabail M, Krishnamoorthy G, Chattopadhyay A.
Journal: J Phys Chem B (2011): 11439
A homogeneous single-label time-resolved fluorescence cAMP assay
Authors: Martikkala E, Rozw and owicz-Jansen A, Hanninen P, Petaja-Repo U, Harma H.
Journal: J Biomol Screen (2011): 356
Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies
Authors: Gaborit N, Larbouret C, Vallaghe J, Peyrusson F, Bascoul-Mollevi C, Crapez E, Azria D, Chardes T, Poul MA, Mathis G, Bazin H, Pelegrin A.
Journal: J Biol Chem (2011): 11337
Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a
Authors: Leyris JP, Roux T, Trinquet E, Verdie P, Fehrentz JA, Oueslati N, Douzon S, Bourrier E, Lamarque L, Gagne D, Galleyr and JC, M'Kadmi C, Martinez J, Mary S, Baneres JL, Marie J.
Journal: Anal Biochem (2011): 253
A time-resolved fluorescence-resonance energy transfer assay for identifying inhibitors of hepatitis C virus core dimerization
Authors: Kota S, Scampavia L, Spicer T, Beeler AB, Takahashi V, Snyder JK, Porco JA, Hodder P, Strosberg AD.
Journal: Assay Drug Dev Technol (2010): 96
Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs
Authors: Visser AJ, Laptenok SP, Visser NV, van Hoek A, Birch DJ, Brochon JC, Borst JW.
Journal: Eur Biophys J (2010): 241
Steady-state and time-resolved fluorescence quenching with transition metal ions as short-distance probes for protein conformation
Authors: Posokhov YO, Kyrychenko A, Ladokhin AS.
Journal: Anal Biochem (2010): 284
Ligand regulation of the quaternary organization of cell surface M3 muscarinic acetylcholine receptors analyzed by fluorescence resonance energy transfer (FRET) imaging and homogeneous time-resolved FRET
Authors: Alvarez-Curto E, Ward RJ, Pediani JD, Milligan G.
Journal: J Biol Chem (2010): 23318