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AAT Bioquest

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
  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. 

Spectrum

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® 488 succinimidyl ester4915167500010.910.210.11
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 594 succinimidyl ester58760320000010.5310.050.04
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 710 succinimidyl ester71673915000010.6010.120.07
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester82285025000010.110.16
iFluor® 860 succinimidyl ester85387825000010.10.14
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 560 succinimidyl ester56057112000010.5710.04820.069
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 440 succinimidyl ester4344804000010.6710.3520.229
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74076422500010.2010.160.16
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 770 succinimidyl ester77779725000010.160.090.08
iFluor® 570 succinimidyl ester55757012000010.581--
iFluor® 830 succinimidyl ester830867----
iFluor® 675 succinimidyl ester683700---0.066
iFluor® 620 succinimidyl ester621636---0.04
iFluor® 605 succinimidyl ester603623----
iFluor® 625 succinimidyl ester624640----
iFluor® 510 succinimidyl ester511530----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520----
Show More (36)

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 October 11, 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-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501