Rhodamines

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®). Compared to cyanines, coumarins and Rhodamines, rhodamines are generally more photostable and are pH insensitive. Rhodamine conjugates must be carefully prepared because they are particularly susceptible to quenching when more than two or three dye molecules are covalently attached to a target molecule such as an antibody molecule.

Spectral Properties

Table 1. Spectral properties of tetramethylrhodamine (TRITC).

SpecificationValue
Laser Line:488/532 nm laser
Filter Set:TRITC
Ex (nm):552 nm
Em (nm):578 nm
Extinction Coefficient (ε):85,000 cm-1M-1
Quantum Yield (Φ):0.1
Figure 1. Excitation and emission spectra of tetramethylrhodamine (TRITC).

iFluor™ 555 – a superior alternative to Cy3®, TRITC and Alexa Fluor® 555

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.

Figure 2. Jurakt cells stained with Annexin V-iFluor™ 555 conjugate.

Table 2. Available iFluor™ 555 Reactive Dyes for Labeling Biomolecules.

Product Chemical Reactivity Reactive Moiety Ex (nm) Em (nm) ε1 Unit Size Cat No.
iFluor™ 555 succinimidyl ester Amine Succinimidyl Ester 557 570 100000 1 mg 1028
iFluor™ 555 maleimide Thiol Maleimide 557 570 100000 1 mg 1063
iFluor™ 555 alkyne Azide Alkyne 557 570 100000 1 mg 1092
iFluor™ 555 amine Aldehyde, Carboxylic Acid, Ketone Amine 557 570 100000 1 mg 1073
iFluor™ 555 azide Alkyne Azide 557 570 100000 1 mg 1093
iFluor™ 555 hydrazide Aldehyde, Carboxylic Acid, Ketone Hydrazide 557 570 100000 1 mg 1083

Note

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

 

Rhodamine Products


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.

Reference Standards for PCR

 

Reference Standards for Bioconjugation

 

Rhodamine Precursors

DNA Sequencing Building-Blocks

  • 5-dR110 [5-Carboxy-4,7-dichlororhodamine 110]
  • 5-dROX [5-Carboxy-4,7-dichloror-X-hodamine]
  • 6-dR6G [6-Carboxy-4,7-dichlororhodamine 6G]
  • 6-dROX [6-Carboxy-4,7-dichloror-X-hodamine]
  • 5-dR6G [5-Carboxy-4,7-dichlororhodamine 6G]
  • 5-dTMR [5-Carboxy-4,7-dichlorortetramethylrhodamine]
  • 6-dR110 [6-Carboxy-4,7-dichlororhodamine 110]
  • 6-dTMR [6-Carboxy-4,7-dichlorortetramethylrhodamine]
  • 5-dR110, succinimidyl ester
  • 5-dR6G, succinimidyl ester
  • 5-dROX, succinimidyl ester
  • 5-dTMR, succinimidyl ester

Rhodamine Reactive Dyes


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

 

Table 3. Available Reactive Rhodamine and Rhodamine Derivatives for Labeling Biomolecules.

Product Chemical Reactivity Reactive Moiety Ex (nm) Em (nm) ε1 Unit Size Cat No.
5-CR110 *Single isomer* Amines after pre-activation with carbodiimides Carboxylic Acid 499 525 84000 5 mg 322
6-CR110 *Single isomer* Amines after pre-activation with carbodiimides Carboxylic Acid 499 525 84000 5 mg 323
5(6)-CR6G, SE *Mixed isomers* Amine Succinimidyl Ester 522 546 94000 10 mg 340
5(6)-CR6G, carboxylic acid Amines after pre-activation with carbodiimides Carboxylic Acid 522 546 94000 10 mg 340
5(6)-Carboxyrhodamine 6G cadaverine Labels proteins by transaminidation Cadaverine 522 546 94000 1 mg 343
5(6)-Carboxyrhodamine 6G ethylenediamine Aldehyde, Carboxylic Acid, Ketone Ethylenediamine 522 546 94000 1 mg 344
5-CR6G, SE *Single isomers* Amine Succinimidyl Ester 522 546 94000 5 mg 345
5-CR6G, carboxylic acid *Single isomers* Amines after pre-activation with carbodiimides Carboxylic Acid 522 546 94000 10 mg 331
6-CR6G, SE *Single isomers* Amine Succinimidyl Ester 522 546 94000 1 mg 342
6-CR6G, carboxylic acid *Single isomers* Amines after pre-activation with carbodiimides Carboxylic Acid 522 546 94000 10 mg 332
5(6)-TAMRA, SE *CAS 246256-50-8* Amine Succinimidyl Ester 552 578 90000 25 mg 370
5(6)-TAMRA, Maleimide *Mixed isomers* Thiol Maleimide 552 578 90000 5 mg 412
5(6)-TAMRA, carboxylic acid *CAS 98181-63-6* Amines after pre-activation with carbodiimides Carboxylic Acid 552 578 90000 100 mg 360
5(6)-TAMRA, ethylenediamine Aldehyde, Carboxylic Acid, Ketone Ethylenediamine 552 578 90000 25 mg 354
5-TAMRA, SE *CAS 150810-68-7* Amine Succinimidyl Ester 552 578 90000 5 mg 373
5-TAMRA, azide Alkyne Azide 552 578 90000 5 mg 486
5-TAMRA, alkyne Azide Alkyne 552 578 90000 5 mg 487
5-TAMRA, Maleimide *CAS 154480-30-5* Thiol Maleimide 552 578 90000 1 mg 421
5-TAMRA, carboxylic acid *CAS 91809-66-4* Amines after pre-activation with carbodiimides Carboxylic Acid 552 578 90000 10 mg 363
5-TAMRA, ethylenediamine Aldehyde, Carboxylic Acid, Ketone Ethylenediamine 552 578 90000 5 mg 358
6-TAMRA, SE *CAS 150810-69-8** Amine Succinimidyl Ester 552 578 90000 5 mg 376
6-TAMRA, azide Alkyne Azide 552 578 90000 5 mg 490
6-TAMRA, alkyne Azide Alkyne 552 578 90000 5 mg 491
6-TAMRA, Maleimide *CAS 174568-68-4* Thiol Maleimide 552 578 90000 1 mg 419
6-TAMRA, carboxylic acid *CAS 91809-67-5* Amines after pre-activation with carbodiimides Carboxylic Acid 552 578 90000 10 mg 366
6-TAMRA, ethylenediamine Aldehyde, Carboxylic Acid, Ketone Ethylenediamine 552 578 90000 5 mg 359
6-TAMRA CPG *1000 Å* Oligonucleotide 3’ end labeling 552 578 90000 5 mg 358
5(6)-ROX, SE *Mixed isomers* Amine Succinimidyl Ester 578 604 82000 25 mg 390
5(6)-ROX, carboxylic acid *CAS 198978-94-8* Amines after pre-activation with carbodiimides Carboxylic Acid 578 604 82000 100 mg 380
5-ROX, SE *CAS 209734-74-7* Amine Succinimidyl Ester 578 604 82000 25 mg 389
5-ROX, carboxylic acid *CAS 216699-35-3* Amines after pre-activation with carbodiimides Carboxylic Acid 578 604 82000 100 mg 381
6-ROX, SE *CAS 216699-36-4* Amine Succinimidyl Ester 578 604 82000 5 mg 392
6-ROX, azide Alkyne Azide 578 604 82000 5 mg 494
6-ROX, alkyne Azide Alkyne 578 604 82000 5 mg 495
6-ROX C2 Maleimide Thiol Maleimide 578 604 82000 1 mg 419
6-ROX, carboxylic acid *CAS 216699-35-3* Amines after pre-activation with carbodiimides Carboxylic Acid 578 604 82000 25 mg 382
6-ROXtra™ SE Amine Succinimidyl Ester 578 594 82000 25 mg 382
Bodi Fluor™ R6G NHS Ester [equivalent to Bodipy® R6G, NHS Ester] Amine Succinimidyl Ester 528 547 116000 5 mg 703
LRB Red™ SE Amine Succinimidyl Ester 558 575 10 mg 471
Lissamine Rhodamine B Sulfonyl Chloride *CAS 62796-29-6* Amine Sulfonyl Chloride 558 575 100 mg 470
Texas Red-X, succinimidyl ester *Mixed isomers* *CAS 216972-99-5* Amine Succinimidyl Ester 586 603 116000 5 mg 475
Texas Red-X, succinimidyl ester *Single isomer* *CAS 199745-67-0* Amine Succinimidyl Ester 586 603 116000 5 mg 474
Sulforhodamine 101 sulfonyl chloride [Texas Red®] Amine Sulfonyl Chloride 586 603 116000 10 mg 480
Texas Red® hydrazide Aldehyde, Carboxylic Acid, Ketone Hydrazide 586 603 116000 5 mg 481
Texas Red® maleimide Thiol Maleimide 586 603 116000 5 mg 483
Texas Red® azide Alkyne Azide 586 603 116000 5 mg 484
Texas Red® cadaverine Labels proteins by transaminidation Cadaverine 586 603 116000 5 mg 482
Texas Red® alkyne Azide Alkyne 586 603 116000 5 mg 485

Note

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

 

Rhodamine-Labeled Enzyme Substrates


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.

 

Table 4. Available Fluorescein-Labeled Fluorogenic Substrates.

Product Biological Function Ex (nm) Em (nm) Unit Size Cat No.
FDG [Fluorescein di-beta-D-galactopyranoside] β-galactosidase substrate 498 517 1 g 14001
FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2* Phosphatase Substrate 498 517 5 mg 11600
FCB [Fluorescein di-beta-D-cellobioside] Cellulase (e.g. β-glucosidases, endoglucanases and exoglucanases) Substrate 498 517 1 mg 14025
Casein, FITC-conjugated Generic Protease Substrate 498 517 5 mg 13440
FAM-VAD-FMK Polycaspase Inhibitor (Caspases 1, 2, 3, 6, 8, 9, or 10) 493 517 25 Tests 13470
FITC-C6-DEVD-FMK Caspase 3 Substrate 491 516 100 µg 13408
FITC-C6-D(OMe)E(OMe)VD(OMe)-FMK Caspase 3 Substrate 491 516 100 µg 13417
FITC-C6-LEHD-FMK Caspase 9 Substrate 491 516 100 µg 13409
FDGlcU [Fluorescein di-beta-D-glucuronide] β-Glucuronidase Substrate 498 517 1 mg 14002
Glucose-UDP-Fluorescein Conjugate Glucosyltransferase Substrate 498 517 100 µg 11705
Glucose-UDP-(PEG)6-Fluorescein Conjugate Glucosyltransferase Substrate 498 517 100 µg 11706

Rhodamine-Labeled Conjugates


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.

 

Table 5. Available Rhodamine-Labeled Fluorogenic Conjugates.

Product Biological Function Ex (nm) Em (nm) Unit Size Cat No.
TRITC Goat Anti-human IgG (H+L) Antibody Indirectly detects human primary antibodies 544 570 1 mg 50209
TRITC Goat Anti-human IgG (H+L) Antibody *Cross Adsorbed* Indirectly detects human primary antibodies 544 570 1 mg 50211
Phalloidin-Tetramethylrhodamine Conjugate Labels actin filaments (F-actin) 552 578 300 Tests 23102
Annexin V, TRITC Labeled Detects phosphatidylserine on the cell surface 544 570 100 Tests 20031
TRITC–Dextran Conjugate (average MW = ~20K) Cardiovascular, Microcirculation, Perfusion, Cell Membrane Flux Tracer 544 570 25 mg 21706
TRITC–Dextran Conjugate (average MW = ~70K) Cardiovascular, Microcirculation, Perfusion, Cell Membrane Flux Tracer 544 570 25 mg 21708
TRITC–Dextran Conjugate (average MW = ~150K) Cardiovascular, Microcirculation, Perfusion, Cell Membrane Flux Tracer 544 570 25 mg 21710
Texas Red®-streptavidin conjugate Cardiovascular, Microcirculation, Perfusion, Cell Membrane Flux Tracer 586 603 1 mg 16911
Texas Red® goat anti-mouse IgG (H+L) Indirectly detects mouse primary antibodies 586 603 1 mg 16853
Texas Red® goat anti-mouse IgG (H+L) *Cross Adsorbed* Indirectly detects mouse primary antibodies 586 603 1 mg 16861
Texas Red® goat anti-rabbit IgG (H+L) Indirectly detects rabbit primary antibodies 586 603 1 mg 16869
Texas Red® goat anti-rabbit IgG (H+L) *Cross Adsorbed* Indirectly detects rabbit primary antibodies 586 603 1 mg 16877
PE/Texas Red® goat anti-human IgG (H+L) Indirectly detects human primary antibodies 586 603 1 mg 50249
PE/Texas Red® goat anti-human IgG (H+L) *Cross Adsorbed* Indirectly detects human primary antibodies 586 603 1 mg 50251

Rhodamine-Derived Indicators


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.

 

Table 6. Available Rhodamine-Derived Indicators.

Product Biological Function Ex (nm) Em (nm) Unit Size Cat No.
Rhodamine 123 *CAS 62669-70-9* Live Cell Mitochondria Indicator 508 528 25 mg 22210
Dihydrorhodamine 123 *CAS 109244-58-8* Redox Indicator 508 528 10 mg 15206
Rhodamine B, hexyl ester, perchlorate Live Cell Mitochondria Indicator 546 568 10 mg 22211
TMRM [Tetramethylrhodamine methyl ester] *CAS#: 115532-50-8* Mitochondrial Membrane Potential Indicator 552 574 25 mg 22221
TMRE [Tetramethylrhodamine ethyl ester] *CAS#: 115532-52-0* Mitochondrial Membrane Potential Indicator 552 574 25 mg 22220
TRITC-DHPE Phospholipid Tracer 544 570 1 mg 23301
Texas Red-DHPE *CAS 187099-99-6* Phospholipid Tracer 586 603 1 mg 23300
Rhod-2, AM *UltraPure Grade* *CAS 145037-81-6* Cell-Permeable Calcium Indicator (Kd = 570 nM) 553 577 1 mg 21062
Rhod-2, trisodium salt Cell-Impermeable Calcium Indicator (Kd = 570 nM) 553 577 1 mg 21068
Rhod-2, tripotassium salt Cell-Impermeable Calcium Indicator (Kd = 570 nM) 553 577 1 mg 21067
Rhod-2 dextran conjugate *Low affinity with MW 10,000* Cell-Impermeable Calcium Indicator (Kd = 3.8 µM) 553 577 1 mg 20451
Rhod-4™, AM Cell-Permeable Calcium Indicator (Kd = 451 nM) 523 551 1 mg 21120
Rhod-4™, sodium salt Cell-Impermeable Calcium Indicator (Kd = 451 nM) 523 551 1 mg 21118
Rhod-4™, potassium salt Cell-Impermeable Calcium Indicator (Kd = 451 nM) 523 551 1 mg 21119
Rhod-5N, AM Cell-Permeable Calcium Indicator (Kd = 320 µM) 557 580 1 mg 21070
Rhod-5N, tripotassium salt Cell-Impermeable Calcium Indicator (Kd = 320 µM) 557 580 1 mg 21072
Rhod-FF, AM Cell-Permeable Calcium Indicator (Kd = 19 µM) 553 577 1 mg 21077
Rhod-FF, tripotassium salt Cell-Impermeable Calcium Indicator (Kd = 19 µM) 553 577 1 mg 21075

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