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trFluor™ Tb maleimide

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™ Tb probes enable time-resolved fluorometry (TRF) for the assays that require high sensitivity. These trFluor™ Tb 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™ Tb probes have relatively high stability, high emission yield and ability to be linked to biomolecules. Moreover, our trFluor™ Tb probes are insensitive to fluorescence quenching when conjugated to biological polymers such as 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

trFluor™ Tb Maleimide Stock Solution (Solution B)
  1. Add anhydrous DMSO into the vial of trFluor™ Tb maleimide to make a 10 mM stock solution. Mix well by pipetting or vortex.

    Note: For optimal results, prepare the dye stock solution (Solution B) before starting the conjugation process. Remember to use it promptly, as extended storage of the dye stock solution may reduce its reactivity. Solution B can be stored in the freezer for up to 4 weeks, protected from light and moisture. Avoid freeze-thaw cycles.

Protein Stock Solution (Solution A)
  1. 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 a 1 mL protein labeling stock solution.

    Note: The pH of the protein labeling stock solution 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, it is recommended that the final protein concentration range between 2-10 mg/mL.

  2. Optional. If your protein does not already contain a free cysteine, treating it with either DTT or TCEP is necessary to generate a thiol group. This process converts a disulfide bond into two free thiol groups. If DTT is used, it is important to remove any excess free DTT by dialysis or gel filtration before conjugating a dye maleimide to the protein. Below is a sample protocol for generating a free thiol group:

    1. Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
    2. To make an IgG solution in 20 mM DTT, add 20 µL of DTT stock per ml of IgG solution while mixing. Let the solution stand at room temperature for 30 minutes without additional mixing (to minimize the reoxidation of cysteines to cystines).
    3. Pass the reduced IgG over a filtration column preequilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
    4. Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
    5. Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol).

      Note: For the best results, IgG solutions should be >4 mg/mL. If the antibody is less than 2 mg/mL, it should be concentrated. Include an extra 10% for losses on the buffer exchange column.

      Note: The reduction can be carried out in almost any buffer from pH 7 to 7.5, e.g., MES, phosphate, or TRIS buffers.

      Note: Steps 3 and 4 can be replaced by dialysis.

SAMPLE EXPERIMENTAL PROTOCOL

This protocol was developed for the labeling of goat anti-mouse IgG with trFluor™ Tb maleimide. Further optimization may be required for your specific proteins.

Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity while the protein conjugates of low dye/protein ratio give reduced sensitivity.

Run Conjugation Reaction
  1. Use a 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) to the vial of the protein solution (95 µL of Solution A) with effective shaking. The protein concentration is ~0.05 mM, assuming the protein concentration is 10 mg/mL and the protein molecular weight is ~200KD. 

    Note: We recommend using a 10:1 molar ratio of Solution B (dye) to Solution A (protein). If the ratio is too low 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 the Sephadex G-25 column according to the manufacturer's instructions. 

  2. Load the reaction mixture (from the "Run Conjugation Reaction" section) 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 should be diluted with staining buffer and aliquoted for multiple uses.

    Note: For longer-term storage, the dye-protein conjugate solution needs to be concentrated or freeze-dried. 

Optional: Characterize the Desired Dye-Protein Conjugate

Determining the Degree of Substitution (DOS) is crucial in characterizing dye-labeled proteins. Lower DOS proteins tend to
have weaker fluorescence, but higher DOS proteins may also have reduced fluorescence. For most antibodies, the optimal DOS is between 2 and 10, depending on the dye and protein properties. For effective labeling, the degree of substitution should be controlled to have 5-8 moles of trFluor™ Tb maleimide to one mole of antibody. The following steps are used to determine the DOS of trFluor™ Tb maleimide-labeled proteins:

  1. Measure absorption—To measure the absorption spectrum of a dye-protein conjugate, the sample concentration should
    be kept between 1 and 10 µM, depending on the dye's extinction coefficient. 

  2. Read OD (absorbance) at 280 nm and dye maximum absorption (ƛmax = 544 nm for trFluor™ Tb dyes). For most spectrophotometers, the sample (from the column fractions) must be diluted with de-ionized water so that the OD values range from 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein, while 544 nm is the maximum absorption of trFluor™ Tb maleimide. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye.

  3. Calculate DOS using our DOS calculator:

    https://www.aatbio.com/tools/degree-of-labeling-calculator 

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of trFluor™ Tb 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 mM63.381 µL316.907 µL633.814 µL3.169 mL6.338 mL
5 mM12.676 µL63.381 µL126.763 µL633.814 µL1.268 mL
10 mM6.338 µL31.691 µL63.381 µL316.907 µL633.814 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

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (260 nm)Correction Factor (280 nm)
trFluor™ Eu maleimide *europium complex*298617210000.9110.777

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
Page updated on December 1, 2024

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Catalog Number1444
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Physical properties

Molecular weight

1577.75

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.942

Correction Factor (280 nm)

0.797

Excitation (nm)

333

Emission (nm)

544

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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.
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.