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

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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.

ReadiLink™ Rapid and xtra Rapid iFluor^® Antibody Labeling Kits

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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™ 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

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iFluor^® Secondary Reagents

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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.

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iFluor^® Styramide™ Signal Amplifying Reagents

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.

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

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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

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.

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

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

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

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).

Ordering Information

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Additional Resources

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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.

1. Alexa Fluor^®is a registered trademark of ThermoFisher.
2. HiLyte™ is a trademark of AnaSpec.