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iFluor® Dyes and Kits

AAT Bioquest iFluor® dyes are a series of highly water-soluble fluorescent dyes for covalently labeling biomolecules, such as proteins and antibodies. Available in a variety of excitation-emission profiles, spanning the UV-visible to near-infrared spectrum, there is an iFluor® dye to match any instrument set-up and many combinations for multiplex detection. Conjugates made with iFluor® dyes exhibit superior brightness and photostability, outperforming Alexa Fluor® conjugates and other spectrally similar conjugates. Use iFluor® products for all your immunofluorescence assays, cell imaging, and flow cytometric applications.



iFluor® Reactive Dyes


iFluor® reactive dyes may be covalently labeled to biomolecules without self-quenching, producing intensely fluorescent conjugates. They are widely used to modify amino acids, peptides, proteins (in particular antibodies), oligonucleotides, nucleic acids, carbohydrates and other biological molecules. iFluor® reactive dye formats include amine-reactive succinimidyl ester, thiol-reactive maleimide, and more.

Available Labeling Chemistries

  • Succinimidyl Esters: for labeling primary amines (-NH₂) on proteins, antibodies, peptides, nucleic acids, and other biomolecules. They are commonly used to prepare bioconjugates for immunochemistry, fluorescence in situ hybridization (FISH), cell tracing, receptor labeling, and fluorescent analog cytochemistry.
  • Maleimides: for labeling thiols or sulfhydryl groups (-SH) to selectively modify a protein at a defined site. Thiol-reactive dyes are often used to prepare fluorescent peptides, proteins, and oligonucleotides for probing biological structures, functions, and interactions.
  • Azides: for labeling ethylene groups via Cu(I)-catalyzed Alknye-Azide (CUAAC) or Cu(I)-free strain-promoted Alkyne-Azide Click Chemistry (SPAAC) reaction.
  • Alkynes: for labeling azide groups via Cu(I)-catalyzed Alknye-Azide (CUAAC) or Cu(I)-free strain-promoted Alkyne-Azide Click Chemistry (SPAAC) reaction.
  • Carboxylic acids: for labeling amines after pre-activation with carbodiimides or for Steglich esterification of alcohols.
  • Amines: for labeling various electrophilic compounds such as activated esters.

The table below shows the range of available iFluor® reactive dyes, their excitation and emission maxima, and their fluorophore equivalents to assist you in selecting the best fluorophore for labeling proteins, antibodies, and other biopolymers, as well as derivatizing low molecular weight molecules.

 

Table 1. Available iFluor® Reactive Dyes for Labeling Biomolecules.

Fluorophore
Ex (nm)
Em (nm)
 
Succinimidyl esters
Maleimides
Azides
Alkynes
Hydrazides
Amines
Tetrazines
TCO
Acids
iFluor® 350345450 •1020•1060  •1080•1070   
iFluor® 405403427 •1021•1053  •1081•1071   
iFluor® 430433498 •1052•1054       
iFluor® 440434480 •1041        
iFluor® 450451502 •1026•1057       
iFluor® 460468493 •1022•1058       
iFluor® 488491516 •1023•1062•1000•999•1082•1072•1014•1005 
iFluor® 514511527 •1024        
iFluor® 532537560 •1025•1061       
iFluor® 546541557 •1048•1059       




ReadiLink™ Rapid iFluor® Antibody Labeling Kits and BSA-compatible ReadiLink™ xtra Rapid iFluor® Antibody Labeling Kits provide a quick and convenient method to label microscale volumes of antibodies with our superior iFluor® dyes. The unique chemistry of ReadiLink™ kits enables researchers to effortlessly label and recover 100% of their antibodies without a purification step. Since ReadiLink™ conjugates are covalently labeled, they are stable for long-term storage. They are ideal for demanding applications, including fluorescent microscopy, multi-color imaging, multiplex flow cytometry, primary detection, IHC, FISH, ELISAs, and more. Also available are ReadiLink™ Rapid and xtra Rapid Antibody Labeling kits for conjugation of mFluor™ dyes, Alexa Fluor® dyes, other fluorescent dyes, biotin, BSA, and KLH.

Key features of ReadiLink™ Rapid Antibody Labeling Kits


ReadiLink™ Rapid Antibody Labeling Kit workflow (figure drawn in BioRender).
  • ReadiLink™ xtra Rapid iFluor® Antibody Labeling Kits are compatible with BSA and other stabilizing proteins
  • Label monoclonal antibodies, polyclonal antibodies, or other proteins
  • Quick and simple two-step labeling protocol, no chemistry or conjugation experience necessary
  • Useful for labeling 100 µg of antibody (two separate labeling reactions of 50 µg antibody)
  • 100% antibody recovery, NO purification of the product is required

Learn More

 

Table 2. ReadiLink™ Rapid iFluor® Antibody Labeling Kits

ReadiLink™ Antibody Labeling Kit
Fluorophore Equivalent
Ex/Em (nm)
Imaging
Flow Cytometry
Cat No.
ReadiLink™ Rapid iFluor® 350AMCA, Alexa Fluor® 350344/448Yes 1220
ReadiLink™ xtra Rapid iFluor® 350AMCA, Alexa Fluor® 350344/448Yes 1950
ReadiLink™ Rapid iFluor® 488FITC, Alexa Fluor® 488491/516YesYes1255
ReadiLink™ xtra Rapid iFluor® 488FITC, Alexa Fluor® 488491/516YesYes1955
ReadiLink™ Rapid iFluor® 555Cy3, Alexa Fluor® 555556/569Yes 1227
ReadiLink™ xtra Rapid iFluor® 555Cy3, Alexa Fluor® 555556/569Yes 1958
ReadiLink™ Rapid iFluor® 594Texas Red®, Alexa Fluor® 594587/603Yes 1230
ReadiLink™ xtra Rapid iFluor® 594Texas Red®, Alexa Fluor® 594587/603Yes 1960
ReadiLink™ Rapid iFluor® 633Cy5, Alexa Fluor® 633638/655YesYes1260
ReadiLink™ Rapid iFluor® 647Cy5, Alexa Fluor® 647654/669YesYes1235



iFluor® Secondary Reagents



Comparison of photobleaching rates of iFluor® 488 goat anti-mouse IgG (Cat No. 16735) versus FITC goat anti-mouse IgG (Cat No. 16852). Images were taken at 120-second intervals on a Keyence X710 fluorescence microscope. TFI represents total fluorescence intensity as calculated by ImageJ software.

iFluor® secondary detection reagents are optimized for use in a wide range of fluorescence-based applications, including cell imaging, flow cytometry, and Western blotting. iFluor® conjugates are produced with the highest quality secondary antibodies and streptavidins, and subjected to rigorous quality control assays. To ensure customers receive products with the greatest sensitivity and selectivity, iFluor® secondary antibodies have been affinity purified and cross-adsorbed, providing higher signal-to-noise ratios with minimal cross-reactivity.

iFluor® Secondary Antibodies

Secondary antibodies conjugated to bright and photostable iFluor® dyes can be used to detect and visualize low, medium, and high-abundance targets in various cell and protein analysis studies. iFluor® secondary antibodies recognize IgG heavy chains and immunoglobulin light chains (e.g., kappa or lambda) from either human, mouse, or rabbit. The advantages associated with iFluor® secondary antibody usage include excellent water solubility, higher signal-to-noise ratios, enhanced sensitivity, multiplexing capabilities, and pH-insensitive fluorescence over a wide molar range.


Learn More
 

Table 3. Available iFluor® secondary antibody conjugates.

Label
Abs (nm)
Ex (nm)
Filter Set
Antibody
Host
Reactivity
Cat No.
iFluor® 350344448DAPIAnti-Human IgG (H&L)GoatHuman50041
iFluor® 405402425DAPIAnti-Human IgG (H&L)GoatHuman50045
iFluor® 430433495FITCAnti-Human IgG (H&L)GoatHuman50049
iFluor® 450451502FITCAnti-Human IgG (H&L)GoatHuman50053
iFluor® 488491516FITCAnti-Human IgG (H&L)GoatHuman50057
iFluor® 514527554TRITCAnti-Human IgG (H&L)GoatHuman50061
iFluor® 532543563TRITCAnti-Human IgG (H&L)GoatHuman50065
iFluor® 546541557TRITCAnti-Human IgG (H&L)GoatHuman50069
iFluor® 555556569TRITCAnti-Human IgG (H&L)GoatHuman50073
iFluor® 560559571TRITCAnti-Human IgG (H&L)GoatHuman50077


iFluor® Styramide™ Signal Amplifying Reagents


Multiplex Styramide™ labeling of FFPE tissue. Tissue was incubated with rabbit anti-EpCAM antibodies and mouse anti-pan-Keratin antibodies and then labeled with reagents in the iFluor&trade 488 PSA™ Imaging Kit with Goat Anti-Rabbit IgG (Cat No. 45205) and iFluor&trade 555 PSA™ Imaging Kit with Goat Anti-Mouse IgG (Cat No. 45220), respectively. Nuclei were labeled with DAPI (Cat No. 17507).

Similar to tyramide signal amplification (TSA), Power Styramide™ Signal Amplification (PSA™) uses the catalytic activity of horseradish peroxidase (HRP) for the covalent deposition and binding of labeled-Styramide™ onto a target protein or nucleic acid sequence in situ. Since the added labeled-Styramide™ are deposited in close proximity of the HRP-target site, there is minimal diffusion-related loss of resolution. Moreover, PSA™ imaging technology can be readily added to any application that allows for the integration of HRP into its protocol. Such applications include IHC, ICC, IF, in situ hybridization, and ELISA.

Key features of iFluor® Styramide™ Reagents

  • Increased detection sensitivity more than 100-fold greater than standard IHC, ICC, IF methods
  • Improved fluorescence signal 10 to 50-fold greater than tyramide (TSA) reagents
  • Higher reactivity of iFluor® Styramide™ radicals allow for faster, efficient, and more robust labeling of styramide conjugates at the HRP-target interaction site
  • Conserve precious antibodies, iFluor® Styramide™ labeling allows the use of significantly less primary antibodies or probe without any sacrifice in detection sensitivity
  • Available as PSA™ imaging kits with poly-HRP, easy-to-use with sufficient reagents for 100 tests

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The table below shows the range of available iFluor® Styramide™ reagents, their excitation and emission maxima, extinction coefficient, and fluorescence quantum yield to assist you in selecting the best labeling reagent for your fluorescence-based imaging.

 

Table 4. iFluor® Styramide™ Reagents For iFluor® Secondary Reagents

iFluor® Styramide™
Ex/Em (nm)
Filter Set
Ext. Coeff.1
FQY2
Unit Size
Price
iFluor® 350 Styramide™345/442DAPI20,0000.95100 Test$310
iFluor® 488 Styramide™491/514FITC75,0000.9100 Test$310
iFluor® 546 Styramide™541/557Cy3/TRITC100,0000.67100 Test$310
iFluor® 555 Styramide™552/567Cy3/TRITC100,0000.64100 Test$310
iFluor® 568 Styramide™568/587Cy3/TRITC100,0000.57100 Test$310
iFluor® 594 Styramide™587/603Cy3/TRITC180,0000.53100 Test$310
iFluor® 647 Styramide™654/669Cy5250,0000.25100 Test$310
iFluor® 680 Styramide™683/700Cy5220,0000.23100 Test$310
iFluor® 700 Styramide™690/713Cy7220,0000.23100 Test$310
iFluor® 750 Styramide™759/777Cy7275,0000.12100 Test$310
  1. Ext. Coeff. = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).



iFluor® Bioconjugates


We offer a large selection of iFluor®-labeled conjugates for a wide range of applications. Featured conjugates include Annexin V for apoptotic detection and phalloidin for cell structure labeling.

Annexin V iFluor® Conjugates for Apoptosis Detection


Jurkat cells were treated with 1 µM camptothecin. The externalized PS residues, an early-stage indicator of apoptosis, were detected using Annexin V iFluor® 555 conjugates (Cat No. 20072). Apoptotic cells and necrotic cells were stained with Nuclear Green™ DCS1 (Cat No. 17550).

Annexin V conjugated to iFluor® dyes provide a fast and reliable method for detecting externalized PS, a key characteristic in early-stage apoptosis. The superior brightness and photostability of Annexin V iFluor® conjugates are useful for live-cell imaging, immunofluorescence, and flow cytometry. They can be combined with other dyes, such as nucleic acid stains, to accurately assess mixed populations of apoptotic, necrotic, and dead cells.

Key features of Annexin V iFluor® Conjugates

  • iFluor® conjugates generate brighter, more photostable signals
  • Compatible with live cells only
  • Fixable after staining with formaldehyde
  • Suitable for all fluorescence-based imaging platforms
  • Conjugates to match any laser setting
  • Available in multicolor wavelengths for multiplexing applications
  • Available as stand-alone reagents or easy-to-use kits

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The table below shows the specifications of available annexin V iFluor® conjugates to assist you in selecting the best annexin v conjugate for your fluorescence-based imaging or flow cytometry experiment.

 

Table 5. Available Annexin V iFluor® Conjugates for Apoptosis Detection.

Annexin V Conjugate
Ex (nm)
Em (nm)
Filter Set
ε¹
Φ²
Unit Size
Cat No.
iFluor® 350344344/448DAPI200000.95100 tests (200 µL)20070
iFluor® 488491516FITC750000.9100 tests (200 µL)20071
iFluor® 555556569TRITC1000000.64100 tests (200 µL)20072
iFluor® 594587603Texas Red1800000.53100 tests (200 µL)20073
iFluor® 633638652Texas Red2500000.29100 tests (200 µL)20069
iFluor® 647654669Cy52500000.29100 tests (200 µL)20074
iFluor® 680683700Cy5.52200000.23100 tests (200 µL)20075
iFluor® 700690713Cy5.52200000.23100 tests (200 µL)20077
iFluor® 750756777Cy72750000.12100 tests (200 µL)20076
  1. Ext. Coeff. = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).


Phalloidin iFluor® Conjugates for Staining F-Actin


F-actin of fixed and permeabilized HeLa cells visualized with Phalloidin iFluor® 594 (Cat No. 23122). Endoplasmic reticulum (ER) and nuclei were stained, respectively, with Cell Navigator® Live Cell ER Staining Kit (Cat No. 22635) and DAPI (Cat No. 17507). ER was stained before fixation.

Used at nanomolar concentration, these iFluor® phalloidin derivatives are convenient for many actin-related assays. Such assays include labeling, identifying, and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell-cultures, or cell-free experiments. Compared to traditional fluorescein isothiocyanate (FITC) and rhodamine conjugates, Phalloidin-iFluor® conjugates offer F-actin stains that are superior in brightness and photostability. Phalloidin-iFluor® conjugates are equivalent, and in most cases superior, to Alexa Flour® conjugates in performance.

Key features of Phalloidin iFluor® Conjugates

  • Selectively targets and binds to F-actin, does not bind to monomeric G-actin
  • Compatible with formaldehyde-fixed samples and permeabilized tissue sections, cell cultures, cell-free experiments, and paraffin-embedded samples that have been de-paraffinized
  • Negligible non-specific staining for high-contrast discrimination of actin
  • Available in multicolor wavelengths for multiplexing applications
  • Suitable for fluorescence-based imaging platforms

Learn More

The table below lists specifications for Phalloidin-iFluor® conjugates, their absorbance and emission maxima, extinction coefficient, and fluorescence quantum yield.

 

Table 6. Phalloidin iFluor® Conjugates For iFluor® Bioconjugates

Conjugate
Mol. Wt.
Ex/ Em (nm)
Filter Set
Extinction Coefficient¹
Quantum Yield
Unit Size
Price
Phalloidin-iFluor® 350∼1300344/448DAPI20,0000.95300 Tests$153
Phalloidin-iFluor® 405∼1400402/425DAPI37,0000.91300 Tests$153
Phalloidin-iFluor® 488∼1900491/516FITC75,0000.9300 Tests$153
Phalloidin-iFluor® 514∼1800527/554TRITC80,0000.83300 Tests$153
Phalloidin-iFluor® 532∼1800543/563TRITC90,0000.68300 Tests$153
Phalloidin-iFluor® 555∼1300556/569TRITC100,0000.64300 Tests$153
Phalloidin-iFluor® 594∼1600587/603Texas Red180,0000.53300 Tests$153
Phalloidin-iFluor® 6331703.99638/652Texas Red250,0000.29300 Tests$153
Phalloidin-iFluor® 6471632.87654/669Cy5250,0000.25300 Tests$153
Phalloidin-iFluor® 680∼2000683/700Cy5.5220,0000.23300 Tests$310
  1. Ext. Coeff. = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).



Additional Resources



Table 7. iFluor® Dyes Spectral Properties For Additional Resources

iFluor® Dye
Mol. Wt.
Abs. (nm)
Em. (nm)
Spectrum
ε¹
Φ²
CF at 260 nm³
CF at 280 nm⁴
iFluor® 350749.8534545020,0000.950.830.23
iFluor® 405755.5840342737,0000.910.480.77
iFluor® 430688.7943349840,0000.780.680.3
iFluor® 440692.8343448040,000N/D0.3520.229
iFluor® 450603.6945150240,0000.820.450.27
iFluor® 460793.9946849380,0000.80.980.46
iFluor® 488945.0749151675,0000.90.210.11
iFluor® 5141013.9551152775,0000.830.2650.116
iFluor® 532914.0653756090,0000.680.260.16
iFluor® 5461145.41541557100,0000.670.250.15
  1. Ext. Coeff. = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).
  3. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligo and nucleic acid labeling).
  4. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).
  5. Fluorescence intensity is significantly increased upon coupling to proteins.

Table 8. iFluor® Dyes Equivalents For Additional Resources

If you are using
Try this iFluor® Dye
Alexa Fluor® 350, AMCA, coumariniFluor® 350
Alexa Fluor® 488, Atto 488, HiLyte™ 488, Cy2, FITCiFluor® 488
Alexa Fluor® 555, Atto 550, HiLyte™ 555, Cy3, TRITCiFluor® 555
Alexa Fluor® 568, Atto 565, Rhodamine RediFluor® 568
Alexa Fluor® 594, Atto 594, Texas RediFluor® 594
Alexa Fluor® 647, Atto 647, HiLyte™ 647, Cy5iFluor® 647
Alexa Fluor® 680, Atto 680 Cy5.5, IRDye® 680iFluor® 680
Alexa Fluor® 750, HiLyte™ 750, Cy7iFluor® 750
  1. Alexa Fluor®is a registered trademark of ThermoFisher.
  2. HiLyte™ is a trademark of AnaSpec.

iFluor® Dye Custom Synthesis Service

AAT Bioquest Custom Synthesis service offers a wide selection of labels, including iFluor® dyes, traditional fluorophores, hapten molecules, and enzymes, along with a choice of labeling chemistries to satisfy any research need. For custom synthesis services, please send all inquiries or requests to info@aatbio.com.



Fluorescence Spectrum Viewer


Need assistance selecting the best fluorophore for your experiment, use our Fluorescence Spectrum Viewer:
  • View and compare fluorophores and fluorescent proteins for biological applications
  • Check spectral compatibility
  • Add multiple excitation and emission filters
  • Save spectra configuration as a PNG or hyperlink