iFluor® 555 maleimide

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	961
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.23
Correction Factor (280 nm)	0.14
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	557
Emission (nm)	570
Quantum yield	0.64¹

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

Alternative formats

Overview SDS Protocol

Molecular weight

961

Correction Factor (260 nm)

0.23

Correction Factor (280 nm)

0.14

Extinction coefficient (cm ^-1 M ^-1)

100000¹

Excitation (nm)

557

Emission (nm)

570

Quantum yield

0.64¹

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® 555 dyes have fluorescence excitation and emission maxima of ~557 nm and ~570 nm respectively. The iFluor® 555 family has spectral properties essentially identical to those of Cy3® (Cy3® is the trademark of GE Healthcare). Compared to Cy3 probes, the iFluor® 555 family has much stronger fluorescence and higher photostability. Their fluorescence is pH-independent from pH 3 to 11. These spectral characteristics make this new dye family a superior alternative to Cy3®. iFluor® 555 family has become an excellent replacement for Cy3® and Alexa Fluor® 555 labeling dye (Cy3® and Alexa Fluor® are the trademarks of Invitrogen and GE Health Care). iFluor® 555 maleimide is reasonably stable and shows good reactivity and selectivity with the thiol group.

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™ 555 maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 555 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™ 555 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® 555 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	104.058 µL	520.291 µL	1.041 mL	5.203 mL	10.406 mL
5 mM	20.812 µL	104.058 µL	208.117 µL	1.041 mL	2.081 mL
10 mM	10.406 µL	52.029 µL	104.058 µL	520.291 µL	1.041 mL

Molarity calculator

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Spectrum

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Spectral properties

Correction Factor (260 nm)	0.23
Correction Factor (280 nm)	0.14
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	557
Emission (nm)	570
Quantum yield	0.64¹

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® 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	588	604	180000¹	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® 555 Styramide Superior Replacement for Alexa Fluor 555 tyramide and Opal 570	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 555 Tyramide	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 460 maleimide	468	493	80000¹	~0.8¹	0.98	0.46
iFluor® 555 TCO	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 555 Tetrazine	557	570	100000¹	0.64¹	0.23	0.14
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® 780 maleimide	784	808	250000¹	0.16¹	0.13	0.12
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
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Images

Figure 1. HeLa cells were stained with mouse anti-tubulin followed with iFluor^TM 555 goat anti-mouse IgG (H+L) (red); actin filaments were stained with Phalloidin-iFluor^TM 488 conjugate (green); and nuclei were stained with DAPI (blue).

Citations

View all 7 citations: Citation Explorer

Membrane fission during bacterial spore development requires cellular inflation driven by DNA translocation
Authors: Landajuela, Ane and Braun, Martha and Mart{\'\i}nez-Calvo, Alejandro and Rodrigues, Christopher DA and Perez, Carolina Gomis and Doan, Thierry and Rudner, David Z and Wingreen, Ned S and Karatekin, Erdem
Journal: Current Biology (2022): 4186--4200

A new FRET-based platform to track substrate ubiquitination by fluorescence
Authors: Wu, Kenneth and Ching, Kevin and Chong, Robert A and Pan, Zhen-Qiang
Journal: Journal of Biological Chemistry (2021)

Phase separation-mediated TARP/MAGUK complex condensation and AMPA receptor synaptic transmission
Authors: Zeng, Menglong and D{\'\i}az-Alonso, Javier and Ye, Fei and Chen, Xudong and Xu, Jia and Ji, Zeyang and Nicoll, Roger A and Zhang, Mingjie
Journal: Neuron (2019): 529--543

Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769

Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018

Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function
Authors: Piccard, Helene and Berghmans, Nele and Korpos, Eva and Dillen, Chris and Aelst, Ilse Van and Li, S and ra , undefined and Martens, Erik and Liekens, S and ra , undefined and Noppen, Sam and Damme, Jo Van and others, undefined
Journal: International Journal of Cancer (2012): E425--E436

Requirements for FisB-mediated membrane fission during sporulation
Authors: Landajuela, Ane and Braun, Martha and Rodrigues, Christopher DA and Doan, Thierry and Horenkamp, Florian and Andronicos, Anna and Shteyn, Vladimir and Williams, Nathan D and Lin, Chenxiang and Rudner, David Z and others,

References

View all 49 references: Citation Explorer

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
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Visualizing dengue virus through Alexa Fluor labeling
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Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors
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