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AAT Bioquest

iFluor® 555 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, 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

Prepare iFluor® 555 maleimide stock solution
  1. Allow the vial of iFluor® dye maleimide to warm up to room temperature.
  2. Add anhydrous DMSO to the vial to prepare a 10 mM dye stock solution.
  3. Vortex the vial briefly to fully dissolve the dye, and then centrifuge to collect the dye at the bottom of the vial.
  4. Protect all stock solutions from light as much as possible by wrapping containers in aluminum foil.
Prepare antibody or protein solution for labeling
  1. If your protein already contains a thiol group, prepare the protein at 50-100 uM (for example: 5mg/ml BSA is ~75uM) in 50~100 mM MES buffer or buffers of your choice with pH 6.5~7.0.
  2. If labeling with an intact antibody, reduction of disulfide bonds need to be carried out before maleimide reaction. Prepare antibody in 2-10 mg/ml in a suitable buffer with pH 7.0–7.5. A 10-fold molar excess of a reducing agent such as DTT or TCEP is added to the antibody. If DTT is used, it must be removed by dialysis or desalting to a suitable buffer with pH 6.5~7.0 prior to conjugation. If TCEP is used, it is not necessary to remove excess TCEP during conjugation with maleimides, however, removal of TCEP by dialysis or desalting prior to conjugation gives the better labeling efficiency.

    Below is a sample protocol for generating free thiol groups on antibody:
    1. Prepare 2-10mg/ml IgG solution in PBS.
    2. Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water. 
    3. Add 1- 20 µL of DTT stock per ml of IgG solution while mixing. 
    4. Let the solution stand at room temperature for 30 minutes without additional mixing (to minimize the re-oxidation of cysteines to cystines). 
    5. Pass the reduced IgG over a filtration column pre-equilibrated with 50 mM MES buffer (pH=6.5) to remove excess DTT.
    6. Determine the antibody concentrations. This can be done either spectrophotometrically or colorimetrically.
    7. Carry out the conjugation as soon as possible after this step.

    Note: For the best results, IgG solutions should be > 2 mg/mL.

    Note: The reduction can be carried out in almost any buffer from pH 7 to 7.5, e.g., MES, phosphate, or TRIS buffers.

    Note: Steps 5 can be replaced by dialysis.

     
  3. If your protein doesn’t have a free thiol group or disulfide bond to reduce, a thiolation modification need to be carried out before maleimide conjugation (for example:  using 2-Iminothiolane or 2-IT) to introduce sulfhydryl (-SH) groups to the original amino groups on protein.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the labeling IgG with iFluor® Dye maleimide. Further optimization may be required for your specific proteins.

Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity while the protein conjugates of low dye/protein ratio give reduced sensitivity.

Run Conjugation Reaction
  1. Use a 10~20:1 molar ratio of iFluor® dye maleimide : IgG as the starting point. While stirring or vortexing the protein solution, add a volume of dye stock solution to result in a dye: protein molar ratio of 10-20. For example, for 5mg/ml IgG (~33 uM), you would add dye to a final concentration of 0.33-0.66 mM.

    Note: We recommend using a 10:1 molar ratio of dye to protein.  If the ratio is too low 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. Purify the conjugate on a gel filtration column, such as a Sephadex G-25 column or equivalent matrix, or by extensive dialysis at 4°C in an appropriate buffer.

Recommended AAT Desalting Columns:

Volume of Reaction Catalog#
0.6-1.0mL

Cat#60504: PD-10 Column

https://www.aatbio.com/products/readiuse-disposable-pd-10-desalting-column?unit=60504
~0.1mL

Cat#60500: Spin Column

https://www.aatbio.com/products/readiuse-bio-gel-p-6-spin-column?unit=60500 

Optional: Characterize the Desired Dye-Protein Conjugate

Determining the Degree of Substitution (DOS) is crucial in characterizing dye-labeled proteins. Lower DOS proteins tend to have weaker fluorescence, but higher DOS proteins may also have reduced fluorescence. For most antibodies, the optimal DOS is between 2 and 10, depending on the dye and protein properties. For effective labeling, the degree of substitution should be controlled to have 5-8 moles of iFluor® 555 maleimide to one mole of antibody. The following steps are used to determine the DOS of iFluor® 555 maleimide-labeled proteins:

  1. Measure absorption— To measure the absorption spectrum of a dye-protein conjugate, the sample concentration should be kept between 1 and 10 µM (For example: IgG conjugate: 10uM is ~1.5mg/ml), depending on the dye's extinction coefficient. 
  2. Read OD (absorbance) at 280 nm and dye maximum absorption (ƛ max = 557 nm for iFluor® 555 dyes). For most spectrophotometers, the sample (from the column fractions) must be diluted with de-ionized water so that the OD values range from 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein, while 557 nm is the maximum absorption of iFluor® 555 maleimide. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye.
  3. Calculate DOS using our DOS calculator: https://www.aatbio.com/tools/degree-of-labeling-calculator

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 mg0.5 mg1 mg5 mg10 mg
1 mM104.058 µL520.291 µL1.041 mL5.203 mL10.406 mL
5 mM20.812 µL104.058 µL208.117 µL1.041 mL2.081 mL
10 mM10.406 µL52.029 µL104.058 µL520.291 µL1.041 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 maleimide3454502000010.9510.830.23
iFluor® 405 maleimide4034273700010.9110.480.77
iFluor® 430 maleimide4334984000010.7810.680.3
iFluor® 450 maleimide4515024000010.8210.450.27
iFluor® 460 maleimide468493800001~0.810.980.46
iFluor® 488 maleimide4915167500010.910.210.11
iFluor® 510 maleimide511530----
iFluor® 514 maleimide5115277500010.8310.2650.116
iFluor® 532 maleimide5375609000010.6810.260.16
iFluor® 540 maleimide540557---0.105
iFluor® 546 maleimide54155710000010.6710.250.15
iFluor® 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide and Opal 570*55757010000010.6410.230.14
iFluor® 555 TCO55757010000010.6410.230.14
iFluor® 555 Tetrazine55757010000010.6410.230.14
iFluor® 560 maleimide56057112000010.5710.04820.069
iFluor® 568 maleimide56858710000010.5710.340.15
iFluor® 594 maleimide58760320000010.5310.050.04
iFluor® 605 maleimide603623----
iFluor® 625 maleimide624640----
iFluor® 633 maleimide64065425000010.2910.0620.044
iFluor® 647 maleimide65667025000010.2510.030.03
iFluor® 660 maleimide66367825000010.2610.070.08
iFluor® 665 maleimide667692110,00010.2210.120.09
iFluor® 670 maleimide67168220000010.5510.030.033
iFluor® 680 maleimide68470122000010.2310.0970.094
iFluor® 700 maleimide69071322000010.2310.090.04
iFluor® 720 maleimide71674024000010.1410.150.13
iFluor® 750 maleimide75777927500010.1210.0440.039
iFluor® 770 maleimide77779725000010.160.090.08
iFluor® 780 maleimide78480825000010.1610.130.12
iFluor® 790 maleimide78781225000010.1310.10.09
iFluor® 800 maleimide80182025000010.1110.030.08
iFluor® 810 maleimide81182225000010.0510.090.15
iFluor® 820 maleimide82285025000010.110.16
iFluor® 830 maleimide830867----
iFluor® 840 maleimide8368792000001-0.20.09
iFluor® 860 maleimide85387825000010.10.14
Show More (28)

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

References

View all 49 references: Citation Explorer
Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
Journal: Mol Biosyst (2011): 1766
Visualizing dengue virus through Alexa Fluor labeling
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)
Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells
Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
Journal: Biochem Biophys Res Commun (2011): 211
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 JJ, Zhu XL, Ji CF, Jing XH, Bai WZ.
Journal: Zhen Ci Yan Jiu (2011): 262
Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
Authors: Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S.
Journal: J Microbiol Methods (2011): 119
Page updated on May 19, 2025

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Catalog Number1063
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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-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501
HeLa cells were stained with mouse anti-tubulin followed with iFluor<sup>TM</sup>&nbsp;555 goat anti-mouse IgG (H+L) (red); actin filaments were stained with Phalloidin-iFluor<sup>TM</sup> 488 conjugate (green); and nuclei were stained with DAPI&nbsp;(blue).
HeLa cells were stained with mouse anti-tubulin followed with iFluor<sup>TM</sup>&nbsp;555 goat anti-mouse IgG (H+L) (red); actin filaments were stained with Phalloidin-iFluor<sup>TM</sup> 488 conjugate (green); and nuclei were stained with DAPI&nbsp;(blue).
HeLa cells were stained with mouse anti-tubulin followed with iFluor<sup>TM</sup>&nbsp;555 goat anti-mouse IgG (H+L) (red); actin filaments were stained with Phalloidin-iFluor<sup>TM</sup> 488 conjugate (green); and nuclei were stained with DAPI&nbsp;(blue).