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trFluor™ Eu-Cryptate succinimidyl ester

trFluor™ Cryptate Eu succinimidyl ester has the highest stability with good water solubility. Compared to the other commercial Cryptate Eu probes, trFluor™ Cryptate Eu succinimidyl ester only has a single reactive group, eliminating the crosslinking possibility. trFluor™ Eu probes are insensitive to fluorescence quenching when conjugated to biological polymers such as antibodies.
trFluor™ Cryptate Eu succinimidyl ester has the highest stability with good water solubility. Compared to the other commercial Cryptate Eu probes, trFluor™ Cryptate Eu succinimidyl ester only has a single reactive group, eliminating the crosslinking possibility. trFluor™ Eu probes are insensitive to fluorescence quenching when conjugated to biological polymers such as antibodies.
Ordering information
Price ()
Catalog Number1430
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 weight1573.80
SolventDMSO
Spectral properties
Absorbance (nm)305
Extinction coefficient (cm -1 M -1)280001
Excitation (nm)305
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
1573.80
Absorbance (nm)
305
Extinction coefficient (cm -1 M -1)
280001
Excitation (nm)
305
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™ Cryptate Eu succinimidyl ester has the highest stability with good water solubility. Compared to the other commercial Cryptate Eu probes, trFluor™ Cryptate Eu succinimidyl ester only has a single reactive group, eliminating the crosslinking possibility. 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™ Cryptate Eu probes have extremely high stability, high emission yield and ability to selectively label biomolecules. Moreover, our trFluor™ Eu 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.

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. trFluor™ Eu-Cryptate succinimidyl ester stock solution (Solution B)
Add anhydrous DMSO into the vial of trFluor™ Eu-Cryptate succinimidyl ester 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 trFluor™ Eu-Cryptate succinimidyl ester. 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 trFluor™ Eu-Cryptate succinimidyl ester 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 mM63.54 µL317.702 µL635.405 µL3.177 mL6.354 mL
5 mM12.708 µL63.54 µL127.081 µL635.405 µL1.271 mL
10 mM6.354 µL31.77 µL63.54 µL317.702 µL635.405 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


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spectrum

Spectral properties

Absorbance (nm)305
Extinction coefficient (cm -1 M -1)280001
Excitation (nm)305
Emission (nm)617

Citations


View all 2 citations: Citation Explorer
Systemically-delivered biodegradable PLGA alters gut microbiota and induces transcriptomic reprogramming in the liver in an obesity mouse model
Authors: Chaplin, Alice and Gao, Huiyun and Asase, Courteney and Rengasamy, Palanivel and Park, Bongsoo and Skander, Danielle and Bebek, G{\"u}rkan and Rajagopalan, Sanjay and Maiseyeu, Andrei
Journal: Scientific reports (2020): 1--16
Nano-Antagonist Alleviates Inflammation and Allows for MRI of Atherosclerosis
Authors: Mog, Brian and Asase, Courteney and Chaplin, Alice and Gao, Huiyun and Rajagopalan, Sanjay and Maiseyeu, Andrei
Journal: Nanotheranostics (2019): 342

References


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