iFluor® 670 succinimidyl ester

Name: iFluor® 670 succinimidyl ester
Brand: AAT Bioquest
SKU: 1033
Availability: InStock

AAT Bioquest's iFluor® dyes are optimized for labeling proteins, particularly 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 spectral properties nearly identical to those of Cy5B. Compared to Cy5B, iFluor® 670 has a much higher fluorescence quantum yield with greatly enhanced photostability. In addition, the fluorescence of iFluor® 670 is pH-insensitive over a broad range, pH 3-11. These spectral characteristics make this new dye family a 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

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.
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.

Prepare Sephadex G-25 column according to the manufacture instruction.
Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	108.325 µL	541.624 µL	1.083 mL	5.416 mL	10.832 mL
5 mM	21.665 µL	108.325 µL	216.65 µL	1.083 mL	2.166 mL
10 mM	10.832 µL	54.162 µL	108.325 µL	541.624 µL	1.083 mL

Molarity calculator

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Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® 350 succinimidyl ester	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 405 succinimidyl ester	403	427	37000¹	0.91¹	0.48	0.77
iFluor® 488 succinimidyl ester	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 514 succinimidyl ester	511	527	75000¹	0.83¹	0.265	0.116
iFluor® 532 succinimidyl ester	537	560	90000¹	0.68¹	0.26	0.16
iFluor® 555 succinimidyl ester	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 594 succinimidyl ester	587	603	200000¹	0.53¹	0.05	0.04
iFluor® 633 succinimidyl ester	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 647 succinimidyl ester	656	670	250000¹	0.25¹	0.03	0.03
iFluor® 660 succinimidyl ester	663	678	250000¹	0.26¹	0.07	0.08
iFluor® 680 succinimidyl ester	684	701	220000¹	0.23¹	0.097	0.094
iFluor® 700 succinimidyl ester	690	713	220000¹	0.23¹	0.09	0.04
iFluor® 750 succinimidyl ester	757	779	275000¹	0.12¹	0.044	0.039
iFluor® 610 succinimidyl ester	610	628	110000¹	0.85¹	0.32	0.49
iFluor® 710 succinimidyl ester	716	739	150000¹	0.60¹	0.12	0.07
iFluor® 790 succinimidyl ester	787	812	250000¹	0.13¹	0.1	0.09
iFluor® 800 succinimidyl ester	801	820	250000¹	0.11¹	0.03	0.08
iFluor® 810 succinimidyl ester	811	822	250000¹	0.05¹	0.09	0.15
iFluor® 820 succinimidyl ester	822	850	250000¹		0.11	0.16
iFluor® 860 succinimidyl ester	853	878	250000¹		0.1	0.14
iFluor® 546 succinimidyl ester	541	557	100000¹	0.67¹	0.25	0.15
iFluor® 568 succinimidyl ester	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 430 succinimidyl ester	433	498	40000¹	0.78¹	0.68	0.3
iFluor® 450 succinimidyl ester	451	502	40000¹	0.82¹	0.45	0.27
iFluor® 840 succinimidyl ester	836	879	200000¹	-	0.2	0.09
iFluor® 560 succinimidyl ester	560	571	120000¹	0.57¹	0.0482	0.069
iFluor® 460 succinimidyl ester	468	493	80000¹	~0.8¹	0.98	0.46
iFluor® 440 succinimidyl ester	434	480	40000¹	0.67¹	0.352	0.229
iFluor® 665 succinimidyl ester	667	692	110,000¹	0.22¹	0.12	0.09
iFluor® 690 succinimidyl ester	685	704	220000¹	0.30¹	0.09	0.06
iFluor® 720 succinimidyl ester	716	740	240000¹	0.14¹	0.15	0.13
iFluor® 740 succinimidyl ester	740	764	225000¹	0.20¹	0.16	0.16
iFluor® 597 succinimidyl ester	598	618	100000¹	0.7¹	0.335	0.514
iFluor® 770 succinimidyl ester	777	797	250000¹	0.16	0.09	0.08
iFluor® 780 succinimidyl ester	784	808	250000¹	0.16¹	0.13	0.12
iFluor® 570 succinimidyl ester	557	570	120000¹	0.58¹	-	-
iFluor® 830 succinimidyl ester	830	867	-	-	-	-
iFluor® 675 succinimidyl ester	683	700	-	-	-	0.066
iFluor® 620 succinimidyl ester	621	636	-	-	-	0.04
iFluor® 605 succinimidyl ester	603	623	-	-	-	-
iFluor® 625 succinimidyl ester	624	640	-	-	-	-
iFluor® 510 succinimidyl ester	511	530	-	-	-	-
iFluor® 540 succinimidyl ester	540	557	-	-	-	0.105
iFluor® 445 succinimidyl ester	446	558	-	-	-	-
iFluor® 500 succinimidyl ester	501	520	-	-	-	-
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Citations

View all 6 citations: Citation Explorer

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

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

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

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

Page updated on November 21, 2024

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Catalog Number	1033710337151271562
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Physical properties

Molecular weight	923.15
Solvent	DMSO

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 yield	0.55¹