iFluor® 780 succinimidyl ester

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, 355, 405, 488, 555, 633, 638, 647, 660, and 802 nm). iFluor® 780 is an excellent acceptor dye for preparing tandem colors with APC and PE. These iFluor® 780 tandem colors offer a set of unique color profiles for spectral flow cytometry. iFluor® 780 is one of the brightest NIR dyes, and some of its antibody conjugates are significantly brighter than those prepared with IRDyes of similar wavelengths, such as IRDye 800CW. iFluor® 780 succinimidyl ester is an amine-reactive form used to conjugate with amine-containing molecules such as antibodies and peptides.

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™ 780 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 780 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™ 780 SE. You might need further optimization for your particular proteins. 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.

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® 670 succinimidyl ester	671	682	200000¹	0.55¹	0.03	0.033
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® 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	80000¹	-	0.206	0.088
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References

View all 50 references: Citation Explorer

A receptor-binding radiopharmaceutical for imaging of traumatic brain injury in a rodent model: [99mTc]Tc-tilmanocept.
Authors: Chen, Wen and Barback, Christopher V and Wang, Shanshan and Hoh, Carl K and Chang, Eric Y and Hall, David J and Head, Brian P and Vera, David R
Journal: Nuclear medicine and biology (2021): 107-114

Effect of Formalin Fixation for Near-Infrared Fluorescence Imaging with an Antibody-Dye Conjugate in Head and Neck Cancer Patients.
Authors: Kapoor, Shrey and Lu, Guolan and van den Berg, Nynke S and Krishnan, Giri and Pei, Jacqueline and Zhou, Quan and Martin, Brock A and Baik, Fred M and Rosenthal, Eben L and Nishio, Naoki
Journal: Molecular imaging and biology (2021): 270-276

Structurally symmetric near-infrared fluorophore IRDye78-protein complex enables multimodal cancer imaging.
Authors: Yang, Jiang and Zhao, Chunhua and Lim, Jacky and Zhao, Lina and Tourneau, Ryan Le and Zhang, Qize and Dobson, Damien and Joshi, Suhasini and Pang, Jiadong and Zhang, Xiaodong and Pal, Suchetan and Andreou, Chrysafis and Zhang, Hanwen and Kircher, Moritz F and Schmitthenner, Hans
Journal: Theranostics (2021): 2534-2549

Safety and Stability of Antibody-Dye Conjugate in Optical Molecular Imaging.
Authors: Pei, Jacqueline and Juniper, Georgina and van den Berg, Nynke S and Nisho, Naoki and Broadt, Trevor and Welch, Anthony R and Yi, Grace S and Raymundo, Roan C and Chirita, Stefania U and Lu, Guolan and Krishnan, Giri and Lee, Yu-Jin and Kapoor, Shrey and Zhou, Quan and Colevas, A Dimitrios and Lui, Natalie S and Poultsides, George A and Li, Gordon and Zinn, Kurt R and Rosenthal, Eben L
Journal: Molecular imaging and biology (2021): 109-116

Evaluation of Ac-Lys0(IRDye800CW)Tyr3-octreotate as a novel tracer for SSTR2-targeted molecular fluorescence guided surgery in meningioma.
Authors: Dijkstra, Bianca M and de Jong, Marion and Stroet, Marcus C M and Andreae, Fritz and Dulfer, Sebastiaan E and Everts, Marieke and Kruijff, Schelto and Nonnekens, Julie and den Dunnen, Wilfred F A and Kruyt, Frank A E and Groen, Rob J M
Journal: Journal of neuro-oncology (2021): 211-222

Page updated on December 17, 2024

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Physical properties

Molecular weight	1526.90
Solvent	DMSO

Spectral properties

Absorbance (nm)	779
Correction Factor (260 nm)	0.13
Correction Factor (280 nm)	0.12
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	784
Emission (nm)	808
Quantum yield	0.16¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501