iFluor® 780 maleimide

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 dyes have fluorescence excitation and emission maxima of ~784 nm and ~808 nm respectively. These spectral characteristics make them a unique color for fluorescence imaging and flow cytometry applications. 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 maleimide is a thiol-reactive form used to conjugate with thiol-containing molecules such as reduced antibodies, thiol-modified oligos, 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. iFluor™ 780 maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 780 maleimide 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 upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.

2. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 100 mM MES buffer with pH ~6.0) 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 6.5 ± 0.5. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well. 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.
Optional: if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:

Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
Make IgG solution in 20 mM DTT: add 20 µL of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol). Note: IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column. Note: The reduction can be carried out in almost any buffers from pH 7-7.5, e.g., MES, phosphate or TRIS buffers. Note: Steps 3 and 4 can be replaced by dialysis.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with iFluor™ 780 maleimide. 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® 780 maleimide 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	69.451 µL	347.256 µL	694.512 µL	3.473 mL	6.945 mL
5 mM	13.89 µL	69.451 µL	138.902 µL	694.512 µL	1.389 mL
10 mM	6.945 µL	34.726 µL	69.451 µL	347.256 µL	694.512 µL

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 maleimide	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 488 maleimide	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 555 maleimide	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 647 maleimide	656	670	250000¹	0.25¹	0.03	0.03
iFluor® 680 maleimide	684	701	220000¹	0.23¹	0.097	0.094
iFluor® 700 maleimide	690	713	220000¹	0.23¹	0.09	0.04
iFluor® 750 maleimide	757	779	275000¹	0.12¹	0.044	0.039
iFluor® 790 maleimide	787	812	250000¹	0.13¹	0.1	0.09
iFluor® 800 maleimide	801	820	250000¹	0.11¹	0.03	0.08
iFluor® 810 maleimide	811	822	250000¹	0.05¹	0.09	0.15
iFluor® 820 maleimide	822	850	250000¹		0.11	0.16
iFluor® 860 maleimide	853	878	250000¹		0.1	0.14
iFluor® 532 maleimide	537	560	90000¹	0.68¹	0.26	0.16
iFluor® 594 maleimide	587	603	200000¹	0.53¹	0.05	0.04
iFluor® 405 maleimide	403	427	37000¹	0.91¹	0.48	0.77
iFluor® 430 maleimide	433	498	40000¹	0.78¹	0.68	0.3
iFluor® 568 maleimide	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 633 maleimide	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 450 maleimide	451	502	40000¹	0.82¹	0.45	0.27
iFluor® 460 maleimide	468	493	80000¹	~0.8¹	0.98	0.46
iFluor® 665 maleimide	667	692	110,000¹	0.22¹	0.12	0.09
iFluor® 546 maleimide	541	557	100000¹	0.67¹	0.25	0.15
iFluor® 840 maleimide	836	879	200000¹	-	0.2	0.09
iFluor® 770 maleimide	777	797	250000¹	0.16	0.09	0.08
iFluor® 830 maleimide	830	867	-	-	-	-
iFluor® 514 maleimide	511	527	75000¹	0.83¹	0.265	0.116
iFluor® 660 maleimide	663	678	250000¹	0.26¹	0.07	0.08
iFluor® 670 maleimide	671	682	200000¹	0.55¹	0.03	0.033
iFluor® 720 maleimide	716	740	240000¹	0.14¹	0.15	0.13
iFluor® 560 maleimide	560	571	120000¹	0.57¹	0.0482	0.069
PerCP-iFluor® 780	778	806	350000	-	-	-
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References

View all 5 references: Citation Explorer

Fluorescently Labeled Cetuximab-IRDye800 for Guided Surgical Excision of Ameloblastoma: A Proof of Principle Study.
Authors: Morlandt, Anthony B and Moore, Lindsay S and Johnson, Aubrey O and Smith, Caris M and Stevens, Todd M and Warram, Jason M and MacDougall, Mary and Rosenthal, Eben L and Amm, Hope M
Journal: Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons (2020): 1736-1747

Characterization and Predictive Value of Near Infrared 2-Deoxyglucose Optical Imaging in Severe Acute Pancreatitis.
Authors: de Oliveira, Cristiane and Patel, Krutika and Mishra, Vivek and Trivedi, Ram N and Noel, Pawan and Singh, Abhilasha and Yaron, Jordan R and Singh, Vijay P
Journal: PloS one (2016): e0149073

Evaluation of near infrared fluorescent labeling of monoclonal antibodies as a tool for tissue distribution.
Authors: Conner, Kip P and Rock, Brooke M and Kwon, Gayle K and Balthasar, Joseph P and Abuqayyas, Lubna and Wienkers, Larry C and Rock, Dan A
Journal: Drug metabolism and disposition: the biological fate of chemicals (2014): 1906-13

Synthesis and biological evaluation of panitumumab-IRDye800 conjugate as a fluorescence imaging probe for EGFR-expressing cancers.
Authors: Bhattacharyya, Sibaprasad and Patel, Nimit and Wei, Ling and Riffle, Lisa A and Kalen, Joseph D and Hill, G Craig and Jacobs, Paula M and Zinn, Kurt R and Rosenthal, Eben
Journal: MedChemComm (2014): 1337-1346

Identification of forensic samples by using an infrared-based automatic DNA sequencer.
Authors: Ricci, Ugo and Sani, Ilaria and Klintschar, Michael and Cerri, Nicoletta and De Ferrari, Francesco and Giovannucci Uzielli, Maria Luisa
Journal: Croatian medical journal (2003): 299-305

Page updated on November 21, 2024

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Physical properties

Molecular weight	1439.86
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