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trFluor™ Eu maleimide *europium complex*
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.
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:
- 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).
- 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.
- 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.
- 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:
- 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.
- Stir the reaction mixture at room temperature for 2-4 hours in the dark.
- Purify the conjugate using HPLC (ammonium acetate/water and acetonitrile, pH 7.0).
- Collect and pool the desired fractions.
- 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 *europium complex* 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 | 89.529 µL | 447.644 µL | 895.287 µL | 4.476 mL | 8.953 mL |
| 5 mM | 17.906 µL | 89.529 µL | 179.057 µL | 895.287 µL | 1.791 mL |
| 10 mM | 8.953 µL | 44.764 µL | 89.529 µL | 447.644 µL | 895.287 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (260 nm) | Correction Factor (280 nm) |
| trFluor™ Tb maleimide | 333 | 544 | - | 0.942 | 0.797 |
| trFluor™ Eu DBCO *europium complex* | 298 | 617 | 21000 | 0.911 | 0.777 |
Citations
View all 1 citations: Citation Explorer
Biologischer Lichtsammler (LHCII) für Halbleiternanokristalle (Quantum Dots)
Authors: Werwie, Mara
Journal: (2013)
Authors: Werwie, Mara
Journal: (2013)
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
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)
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
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
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
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
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