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iFluor® 670 succinimidyl ester

HeLa cells were incubated with (+ Tubulin) or without (-Tubulin) mouse anti-tubulin followed by iFluor® 670 goat anti-mouse IgG conjugate stain and visualized with Cy5 Filter.
HeLa cells were incubated with (+ Tubulin) or without (-Tubulin) mouse anti-tubulin followed by iFluor® 670 goat anti-mouse IgG conjugate stain and visualized with Cy5 Filter.
Ordering information
Price ()
Catalog Number1033
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 weight923.15
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.033
Extinction coefficient (cm -1 M -1)2000001
Excitation (nm)671
Emission (nm)682
Quantum yield0.551
Storage, safety and handling
Intended useResearch Use Only (RUO)
StorageFreeze (< -15 °C); Minimize light exposure

OverviewpdfSDSpdfProtocol


Molecular weight
923.15
Correction Factor (260 nm)
0.03
Correction Factor (280 nm)
0.033
Extinction coefficient (cm -1 M -1)
2000001
Excitation (nm)
671
Emission (nm)
682
Quantum yield
0.551
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® 670 family has the spectral properties essentially identical to those of Cy5B. Compared to Cy5, iFluor® 670 has much higher fluorescence quantum yield with greatly enhanced photostability. iFluor® 670 family has fluorescence that is pH-independent from pH 3 to 11. These spectral characteristics make this new dye family an superior alternative to Cy5B. iFluor® 670 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™ 670 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 670 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™ 670 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® 670 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 mM108.325 µL541.624 µL1.083 mL5.416 mL10.832 mL
5 mM21.665 µL108.325 µL216.65 µL1.083 mL2.166 mL
10 mM10.832 µL54.162 µL108.325 µL541.624 µL1.083 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)0.03
Correction Factor (280 nm)0.033
Extinction coefficient (cm -1 M -1)2000001
Excitation (nm)671
Emission (nm)682
Quantum yield0.551

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® 570 succinimidyl ester56057112000010.5810.0480.069
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® 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)

Citations


View all 6 citations: Citation Explorer
A combined solvatochromic shift and TDDFT study probing solute-solvent interactions of blue fluorescent Alexa Fluor 350 dye: Evaluation of ground and excited state dipole moments
Authors: Patil, M. K., Kotresh, M. G., Inamdar, S. R.
Journal: Spectrochim Acta A Mol Biomol Spectrosc (2019): 142-152
Photobleaching Comparison of R-Phycoerythrin-Streptavidin and Streptavidin-Alexa Fluor 568 in a Breast Cancer Cell Line
Authors: Ostad, S. N., Babaei, S., Bayat, A. A., Mahmoudian, J.
Journal: Monoclon Antib Immunodiagn Immunother (2019): 25-29
Comparison between photostability of Alexa Fluor 448 and Alexa Fluor 647 with conventional dyes FITC and APC by flow cytometry
Authors: Rai, S., Bhardwaj, U., Misra, A., Singh, S., Gupta, R.
Journal: Int J Lab Hematol (2018): e52-e54
Development of new hCaM-Alexa Fluor((R)) biosensors for a wide range of ligands
Authors: Velazquez-Lopez, I., Leon-Cruz, E., Pardo, J. P., Sosa-Peinado, A., Gonzalez-Andrade, M.
Journal: Anal Biochem (2017): 13-22
Neuroanatomical basis of clinical joint application of "Jinggu" (BL 64, a source-acupoint) and "Dazhong" (KI 4, a Luo-acupoint) in the rat: a double-labeling study of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594
Authors: Cui, J. J., Zhu, X. L., Ji, C. F., Jing, X. H., Bai, W. Z.
Journal: Zhen Ci Yan Jiu (2011): 262-7
Neuroanatomical characteristics of acupoint "Chengshan" (BL 57) in the rat: a cholera toxin subunit B conjugated with Alexa Fluor 488 method study
Authors: Zhu, X. L., Bai, W. Z., Wu, F. D., Jiang, J., Jing, X. H.
Journal: Zhen Ci Yan Jiu (2010): 433-7

References


View all 1 references: Citation Explorer
Improved fluoroimmunoassays using the dye Alexa Fluor 647 with the RAPTOR, a fiber optic biosensor
Authors: Anderson GP, Nerurkar NL.
Journal: J Immunol Methods (2002): 17