iFluor Dyes and Kits

iFluor™ Reactive Dyes

iFluor™ reactive dyes may be covalently labeled to biomolecules without self-quenching, producing intensely fluorescent conjugates. iFluor™ reactive dye formats include amine-reactive succinimidyl ester, thiol-reactive maleimide, and more. For assistance labeling proteins with succinimidyl ester or maleimide iFluor™ dyes, use our online interactive protein labeling protocol.

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

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 enable 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 and 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&reg 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

Figure 1. ReadiLink™ Rapid Antibody Labeling Kit workflow (figure drawn in BioRender).

iFluor™ Secondary Reagents

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 a variety of cell and protein analysis studies. iFluor™ secondary antibodies recognizes IgG heavy chains as well as immunoglobulin light chains (e.g. kappa or lambda) from either human, mouse or rabbit. The advantages of associated with iFluor™ secondary antibody usage includes excellent water solubility, higher signal-to-noise ratios, enhanced sensivitiy, multiplexing capabilities, and pH-insensitive fluorescence over a wide molar range.

Figure 2. 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.

Table 3. Available iFluor™ secondary antibody conjugates.

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 a faster, efficient and more robust labeling of styramide conjugates at the HRP-target interaction site
• Conserve precious antibodies, iFluor™ Styramide™ labeling allows 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

Figure 3. Multiplex Styramide™ labeling of FFPE tissue. Tissue were 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).

The table below shows the range of available iFluor™ Styramide™ reagents, their excitation and emission maximas, 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

We offer a large selection of iFluor™-labeled conjugates for a wide range of applications. Featured conjugates include, Annexin V for apoptic 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, and can be combined with other dyes, such as nucleic acid stains, to accurately assess mixed populations of apoptotic, necortic 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

Figure 4. Jurkat cells were treated with 1 µM camptothecin. The externalized PS residues, an early-stage indicator of apoptosis, was detected using Annexin V iFluor™ 555 conjugates (Cat No. 20072). Apoptotic cells and necrotic cells were were stained with Nuclear Green™ DCS1 (Cat No. 17550).

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.

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 isothiocynanate (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

Figure 5. 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 Stainig Kit (Cat No. 22635) and DAPI (Cat No. 17507). ER was stained before fixation.

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

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