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iFluor® 570 succinimidyl ester

Ordering information
Price ()
Catalog Number1034
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 weight881.07
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.048
Correction Factor (280 nm)0.069
Extinction coefficient (cm -1 M -1)1200001
Excitation (nm)560
Emission (nm)571
Quantum yield0.581
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
881.07
Correction Factor (260 nm)
0.048
Correction Factor (280 nm)
0.069
Extinction coefficient (cm -1 M -1)
1200001
Excitation (nm)
560
Emission (nm)
571
Quantum yield
0.581
AAT Bioquest's iFluor® dyes are optimized for labeling proteins, in particular, antibodies. These dyes are bright, photostable and have minimal quenching on proteins. They can be well excited by the major laser lines of fluorescence instruments (e.g., 350, 405, 488, 555 and 633 nm). iFluor® 570 family has the spectral properties essentially identical to those of Cy3B. It is a superior replacement to Cy3B with greatly improved water solubility compared to Cy3B that is barely soluble in aqueous solutions. It maintains other charactuers of Cy3B, e.g., high fluorescence quantum yield with greatly enhanced photostability. iFluor® 570 family has fluorescence that is pH-independent from pH 3 to 11. iFluor® 570 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups.

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. iFluor™ 570 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 570 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 iFluor™ 570 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 iFluor® 570 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 mM113.498 µL567.492 µL1.135 mL5.675 mL11.35 mL
5 mM22.7 µL113.498 µL226.997 µL1.135 mL2.27 mL
10 mM11.35 µL56.749 µL113.498 µL567.492 µL1.135 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

Correction Factor (260 nm)0.048
Correction Factor (280 nm)0.069
Extinction coefficient (cm -1 M -1)1200001
Excitation (nm)560
Emission (nm)571
Quantum yield0.581

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 succinimidyl ester3454502000010.9510.830.23
iFluor® 405 succinimidyl ester4034273700010.9110.480.77
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 440 succinimidyl ester4344804000010.6710.3520.229
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 488 succinimidyl ester4915167500010.910.210.11
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 560 succinimidyl ester56057112000010.5710.04820.069
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 594 succinimidyl ester58860418000010.5310.050.04
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 710 succinimidyl ester71773919000010.6010.120.07
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74276422500010.2010.160.16
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 770 succinimidyl ester77779725000010.160.090.08
iFluor® 780 succinimidyl ester78480825000010.1610.130.12
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester8228502500001-0.110.16
iFluor® 830 succinimidyl ester830867----
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 860 succinimidyl ester8538782500001-0.10.14
Show More (38)

References


View all 37 references: Citation Explorer
Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labeled Ligands Binding to MC4 Receptors in Budded Baculoviruses.
Authors: Veiksina, Santa and Tahk, Maris-Johanna and Laasfeld, Tõnis and Link, Reet and Kopanchuk, Sergei and Rinken, Ago
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 119-136
Quantitative analysis of fluorescent ligand binding to dopamine D3 receptors using live-cell microscopy.
Authors: Allikalt, Anni and Laasfeld, Tõnis and Ilisson, Mihkel and Kopanchuk, Sergei and Rinken, Ago
Journal: The FEBS journal (2021): 1514-1532
Fluorescent ligands for dopamine D2/D3 receptors.
Authors: Allikalt, Anni and Purkayastha, Nirupam and Flad, Khajidmaa and Schmidt, Maximilian F and Tabor, Alina and Gmeiner, Peter and Hübner, Harald and Weikert, Dorothee
Journal: Scientific reports (2020): 21842
Unifying Mechanism for Thiol-Induced Photoswitching and Photostability of Cyanine Dyes.
Authors: Gidi, Yasser and Payne, Liam and Glembockyte, Viktorija and Michie, Megan S and Schnermann, Martin J and Cosa, Gonzalo
Journal: Journal of the American Chemical Society (2020): 12681-12689
Fluorescence lifetime imaging with a single-photon SPAD array using long overlapping gates: an experimental and theoretical study.
Authors: Ardelean, Andrei and Ulku, Arin Can and Michalet, Xavier and Charbon, Edoardo and Bruschini, Claudio
Journal: Proceedings of SPIE--the International Society for Optical Engineering (2019)
Redox-Based Photostabilizing Agents in Fluorescence Imaging: The Hidden Role of Intersystem Crossing in Geminate Radical Ion Pairs.
Authors: Glembockyte, Viktorija and Cosa, Gonzalo
Journal: Journal of the American Chemical Society (2017): 13227-13233
Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.
Authors: Link, Reet and Veiksina, Santa and Rinken, Ago and Kopanchuk, Sergei
Journal: European journal of pharmacology (2017): 58-66
Predicting signatures of anisotropic resonance energy transfer in dye-functionalized nanoparticles.
Authors: Gil, Gabriel and Corni, Stefano and Delgado, Alain and Bertoni, Andrea and Goldoni, Guido
Journal: RSC advances (2016): 104648-104656
Homogeneous fluorescence anisotropy-based assay for characterization of ligand binding dynamics to GPCRs in budded baculoviruses: the case of Cy3B-NDP-α-MSH binding to MC4 receptors.
Authors: Veiksina, Santa and Kopanchuk, Sergei and Mazina, Olga and Link, Reet and Lille, Anne and Rinken, Ago
Journal: Methods in molecular biology (Clifton, N.J.) (2015): 37-50
Cholesterol-induced lipophobic interaction between transmembrane helices using ensemble and single-molecule fluorescence resonance energy transfer.
Authors: Yano, Yoshiaki and Kondo, Kotaro and Kitani, Ryota and Yamamoto, Arisa and Matsuzaki, Katsumi
Journal: Biochemistry (2015): 1371-9