Rhodamines and Rhodamine Derivatives

Rhodamines are xanthene derivatives structurally related to fluorescein, but with additional chemical substitutions that shift their excitation and emission spectra to longer wavelengths. The most widely used rhodamines include rhodamine 110, the parent rhodamine, tetramethylrhodamine (such as TAMRA or TRITC) and X-rhodamines (such as Texas Red^®). In comparison to coumarins and cyanines, the fluorescence of rhodamines are generally more resistant to photodegradation and insensitive to pH changes. However, rhodamine conjugates are particularly susceptible to self-quenching. When more than two or three fluorophores are covalently attached to a target molecule, such as an antibody or nucleic acid, there is considerable loss in fluorescence.

Spectral Properties

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iFluor® 555 - a superior alternative to Cy3^®, TRITC and Alexa Fluor^® 555

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The iFluor® 555 dye, which is spectrally similar to Cy3^®, TRITC and Alexa Fluor^® 555, produces significantly brighter and more photostable conjugates with pH-insensitive fluorescence over a wide molar range (pH 3 to 11). Ideal for imaging low-abundance targets with greater sensitivity, iFluor® 555 dye molecules can be conjugated to biomolecules, in particular antibodies and proteins, at high molar ratios with minimal self-quenching. These conjugates are well-suited for imaging and are particularly useful in multicolor applications, such as flow cytometry and fluorescence microscopy, without compromising the performance of blue and far-red fluorescent probes.

iFluor 555™ dyes are readily available in a variety of reactive dye forms and protein labeling kits allowing you to easily produce antibody conjugates or probes 'in-house'. Also available are iFluor 555™ dyes conjugated to antibodies, annexin V, phalloidin, amplification substrates (e.g. labeled-tyramides, labeled-styramides) and other biomolecules for cellular imaging and detection assays requiring increased sensitivity.

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1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).

Rhodamine Products

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We offer a broad selection of rhodamine products including reactive rhodamine and rhodamine derivatives for labeling antibodies, nucleic acids and other biomolecules, conjugates, indicators, and substrates for detecting enzymatic activity in cells, homogenates and solution.

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Rhodamine Reactive Dyes

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Reactive fluorescent rhodamine and rhodamine derivatives are widely used to covalently label biomolecules such as antibodies, carbohydrates, nucleic acids, proteins, peptides and oligonucleotides. Resulting in intensely bright and photostable orange fluorescence, rhodamine conjugates are often used as substrates for detecting enzymatic activity or to provide contrast in multicolored fluorescence investigation when combined with blue, green or far-red fluorescent probes. Rhodamine and rhodamine derivatives are readily available in a variety of dye-labeling chemistries, these include:

• Succinimidyl esters/NHS esters - for labeling free amines (-NH₂) on antibodies, proteins, peptides, amine-modified oligonucleotides and other biomolecules
• Maleimides - for labeling sulfhydryl groups on antibodies, proteins and peptides
• Azides - for labeling ethylene groups via click chemistry methods
• Alkynes - for labeling azides via click chemistry methods
• Hydrazides - for labeling aldehyde and ketone groups
• Carboxylic acids - for labeling amines after pre-activation with carbodiimides or for Steglich esterification of alcohols
• Amines - for labeling with various electrophilic compounds such as activated esters
• Tetrazines - for labeling trans-cyclooctenes (TCO) compounds via Inverse-Electron-Demands Diels-Alder reaction
• Phosphoramidite - for labeling oligonucleotides

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).

Rhodamine-Labeled Enzyme Substrates

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Rhodamine and rhodamine derivatives are frequently used to prepare fluorogenic substrates for detecting enzymatic activity in cells and solutions. When hydrolyzed by the enzyme of interest, rhodamine-modified substrates exhibit an increase in fluorescence intensity that is linearly proportional to enzyme activity. AAT Bioquest provides a large assortment of rhodamine modified substrates for detecting activity from caspases, proteases, peptidases, phosphatases and more.

Rhodamine-Labeled Conjugates

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Rhodamine-labeled conjugates have a wide range of applications in bio-imaging and bio-labelling due to their bright orange fluorescence and excellent photostability. Rhodamine derivatives can be labeled to antibodies, streptavidin, phalloidin, annexin v, dextran and other biomolecules to study apoptosis, label organelles or improve assay sensitivity by amplifying weak signals.

Rhodamine-Derived Indicators

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Rhodamine-derived indicators are important tools in biochemical and cellular studies. They have been used in all major types of fluorescence imaging applications including flow cytometry, fluorescence microscopy and fluorescence plate-based assays. AAT Bioquest offers a wide range of rhodamine-derived indicators for measuring cellular pH, calcium concentrations, cell proliferation, cell viability and more.