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AAT Bioquest

Tide Fluor™ and Tide Quencher™ Dyes, Optimized for Maximizing The Power of Fluorescence Resonance Energy Transfer (FRET)

Introduction


Diagram of FRET process
Diagram of FRET process from the donor to acceptor molecule. Horizontal lines represent discrete electron energy levels for each molecule. Energy levels are labeled as either singlet states (S) or triplet states (T) with subscripts numbered zero, one or two (representing the ground state, first excited electronic state or second excited electronic state). A molecule's electrons generally reside in the ground state, S0. Electrons may be excited to higher energy levels by a number of processes, including light absorption and chemical reaction.
Fluorescence resonance energy transfer (FRET) is the transfer of the excited state energy from the initially excited donor (D) to an acceptor (A). The donor molecules typically emit at shorter wavelengths that overlap with the absorption of acceptors. The process is a distance-dependent interaction between the electronic excited states of two molecules without emission of a photon. FRET is the result of long-range dipole-dipole interactions between the donor and acceptor as illustrated in Figure 1. A few excellent recent reviews are listed in the references for further information.

As shown in Figure 1, an excited donor molecule has several routes to release its captured energy returning to the ground state. The excited state energy can be dissipated to the environment (as light or heat) or transferred directly to a second acceptor molecule, sending the acceptor to an excited state. The later process is called FRET.
  • Internal Energy Conversions: The rapid return of electrons from the second (or higher) excited state to the first excited state is termed internal conversion. Energy is released through rapid solvent relaxation.
  • Light Emissions: Light is released by either the transition from S1 to S0 (fluorescence emission) or transition from T1 to S0 (phosphorescence emission).
  • Fluorescence Resonance Energy Transfer: FRET occurs when donor and acceptor molecules are within a specified range, usually within 10 to 100 Å. In the process of FRET, the excited-state energy of a donor is transferred to an acceptor molecule. Photons of light aren't involved in this transfer. In Figure 1, the pathway leading from the S1 level of the donor to the S1 level of the acceptor represents FRET. Once excited, the acceptor can return to the ground state by the same pathways as described for the donor. If the acceptor molecule is also fluorescent, it can emit light at its characteristic wavelength, which is always longer than the emission wavelength of the donor.
  • Collisional Quenchings: Collisions between an excited-state fluorophore and other molecules will sometimes quench the fluorophore, returning it to the ground state without emission of a photon. Collisional quenchers include molecular oxygen and electron scavengers such as Cu2+, Mn2+, halides, isothiocyanate, nitrite and nitrate ions. Collisional quenching primarily occurs when these ions are present in the millimolar range or higher. Therefore, under most experimental conditions, collisional quenching is usually negligible.

 

Optimal FRET Conditions


The efficiency (E%) and rate (kT) of FRET are respectively expressed as follows: E% = kT/(τD-1 + kT) [1]

kT = Ro6 γ-6 τD-1 [2]

Where τD is the decay time of the donor in the absence of acceptor; γ is the donor-acceptor (D-A) distance; Ro is the Förster distance where FRET has 50% efficiency (typically in the range of 20-60Å). Ro is determined by the following equation:

Ro6 = 8.79x1023[k2n-4ΦDJ(λ)] [3]

Where k2 is dipole-dipole orientation factor (ranging from 0 to 4, k2 = 2/3 for randomly oriented donors and acceptors); n is refractive index [The refractive index is generally known from solvent composition or estimated for macromolecules such as proteins and nucleic acids. n is often assumed to be that of water (n=1.33) for aqueous solutions, or to be that of small organic molecules (n=1.39) for organic solutions]. ΦD is the fluorescence quantum yield of donor in the absence of acceptor. J(λ) is FRET spectral overlap integral as illustrated in Figure 2, and is determined by the following equation:

J(λ) = ∫FD(λ)εA(λ)λ4d(λ) [4]

Where FD(λ) is the corrected fluorescence intensity of the donor in the wavelength range λ to λ+∆λ with the total intensity (area under the curve) normalized unity; εA is extinction coefficient of the acceptor at λ.

From the above equations, it is easily concluded that an efficient FRET should have the following conditions:

Schematic
Schematic representation of the FRET spectral overlap integral.
  • Distance between Donor and Acceptor: Donor and acceptor molecules must be in close proximity (typically 10–100 Å).
  • Spectral Overlap: The absorption spectrum of the acceptor must overlap fluorescence emission spectrum of the donor (see Figure 2).
  • Dipole Orientation: Donor and acceptor transition dipole orientations must be approximately parallel.
 

Typical Biological Applications of FRET Probes


Schematic
Schematic representation of fluorogenic Molecular Beacon probes.
As discussed above, the rate and efficiency of FRET are dependent on the inverse sixth power of the intermolecular separation. FRET is highly efficient if the donor and acceptor are positioned within the Förster radius, which is typically 30-60 Å. The distance dependence of FRET has resulted in its widespread biological applications since the distances are comparable with the dimensions of biological macromolecules. For instance, one helical turn in B-DNA spans 33.2 Å. Further, FRET efficiency falls dramatically as the distance between the donor and acceptor exceeds the Förster radius, making FRET an important technique for investigating a variety of biological phenomena in which tracking physical proximity is important. If the donor molecule's fluorescence is quenched, it indicates that the donor and acceptor molecules are within the Förster radius, whereas if the donor fluoresces at its characteristic wavelength, it denotes that the distance between the donor and acceptor molecules has increased beyond the Förster radius. In recent years, FRET-based assays have found broad applications in detecting proteases, real-time assays of hybridization, PCR monitoring and DNA interactions in living cells.

Nucleic Acid Detection


Molecular Beacons, which are 'hairpin' oligonucleotides containing both a fluorescent reporter dye and a quencher as shown in Figure 3, are widely used to detect DNA hybridization. In the absence of target, the fluorescent reporter and quencher molecules are brought close together in the probe's selfcomplementary stem structure, and the fluorescent signal is suppressed. When the Molecular Beacon hybridizes to its target, the fluorescent reporter and the quencher are separated, and the reporter dye emits at its characteristic wavelength. Molecular Beacons have enhanced specificity for their targets, making them superior in hybridizationbased investigations of single nucleotide polymorphisms (SNPs). Molecular Beacons have been used in hybridization-based assays to identify rifampin-resistant Mycobacterium and to detect virus replication in HIV type 1-infected individuals. Molecular Beacons have also been widely used to detect nucleases. Dual-labeled FRET probes are commonly used to detect the 5' exonuclease activity of Taq polymerase. The two labels on the FRET oligonucleotide can be separated by as many as 25-30 bases. If the oligonucleotide is intact, the donor molecule's signal is quenched. During the course of the assay, the cleavage event separates the donor molecule from its acceptor, restoring the donor's characteristic emission. If a dark quencher is used as the acceptor molecule, multiple FRET probes can be used, each labeled with a unique fluorophore, making these probes amenable to multiplex assays.

Hybridization-induced fluorescence
Hybridization-induced fluorescence enhancement of Molecular Beacon oligonucleotide probes that contain Tide Fluor™ dyes as donor or Tide Quencher™ dyes as acceptors.
AAT Bioquest offers a complete set of dye phosphoramidites and dye CPG supports for preparing FRET oligonucleotides including the classic dyes (such as FAM, HEX, TET and JOE) and superior oligo-labeling dyes such as our Tide Fluor™ and Tide Quencher™ dyes. Our Tide Fluor™ dyes (such as TF1, TF2, TF3, TF4, TF5, TF6, TF7 and TF8) have stronger fluorescence and higher photostability than the typical fluorophores such as fluoresceins, rhodamines and cyanines as described below. Our TF2 has the essentially same excitation and emission wavelengths as those of carboxyfluoresceins (FAM), making them readily used for the biological applications that were done with fluoresceins, but have enhanced performance with TF2. Compared to FAM probes, TF2 has much stronger fluorescence at physiological conditions, and it is much more photostable. Compared to other fluorescent dyes alternative to fluoresceins and Cy dyes (such as Alexa Fluor® and Cy3, Cy5 and Cy7), Tide Fluor™ dyes are much more cost-effective with comparable or even better performance for your desired biological applications.

Besides our outstanding Tide Fluor™ donor dyes, AAT Bioquest has also developed the robust Tide Quencher™ acceptor dyes. These Tide Quencher™ dark FRET acceptors (such as TQ1, TQ2, TQ3, TQ4, TQ5, TQ6 and TQ7) are optimized to pair with our Tide Fluor™ dyes and the classic fluorophores (such as AMCA, EDANS, FAM, TAMRA, HEX, JOE, TET, ROX, Cy3, Cy5 and Cy7). Like our Tide Fluor™ donor dyes, our Tide Quencher™ acceptor dyes are much more cost-effective with comparable or even better performance for your desired biological applications than other similar products on the market.

To facility your research, we offer a variety of reactive forms for both our Tide Fluor™ donors and Tide Quencher™ acceptors. For in-synthesis labeling of oligonucleotides, we offer both phosphoramidites of our Tide Fluor™ and Tide Quencher™ dyes and their CPG supports. For post labeling of oligonucleotides, we offer both amino-reactive and thiol-reactive Tide Fluor™ and Tide Quencher™ dyes that are water-soluble.

Protease Detection


Protease cleavage
HIV Protease cleavage of Tide Fluor™ 2/Tide Quencher™ 2-derived FRET peptide. Upon cleavage, the fluorescence of Tide Fluor™ 2 is recovered, and can be monitored at EX/EM = 490/520 nm. With excellent fluorescence quantum yield and longer excitation and emission wavelength, the fluorescence signal of Tide Fluor™ 2 is less interfered by the autofluorescence of cell components and test compounds, thus providing better assay sensitivity.
Proteases are involved in a number of physiological processes. The detection of proteases and the screening of specific protease inhibitors are essential in the discovery of potential drugs for the treatment and management of protease-related diseases. A variety of donor and acceptor molecules (either fluorescent or non-fluorescent) are attached to the peptide/protein (forming the internally quenched conjugates), and are used to detect a protease depending on the sequences of the amino acids. The donor and acceptor molecules are carefully chosen so that the absorption of the acceptor overlaps with the fluorescence of the donor, thus ensuring that the fluorescence is quenched through resonance energy transfer. Enzyme hydrolysis of the substrate results in spatial separation of the donor and the acceptor molecules, thereby restoring the donor's fluorescence. For example, EDANS and DABCYL are used to label a peptide that contains the HIV protease cleavage site. Proteolytic cleavage releases a fragment containing only the EDANS fluorophore, thus liberating it from the quenching effect of the nearby DABCYL chromophore. AAT Bioquest offers a variety of reagents for developing FRET-based protease probes and assays. Please visit our websites at www.aatbio.com for protease assays or www.bioconjugation.com for FRET probe building blocks.

HIV protease has been identified as one of the key targets for the development of anti-AIDS drugs. The aspartic HIV protease, a 10-12 kD of human immunodeficiency virus-1 (HIV-1), is required for the posttranslational cleavage of the precursor polyproteins, Prgag and Prgag-pol. These cleavages are essential for the maturation of HIV infectious particles. A Tide Fluor™ 2/Tide Quencher™ 2-derived FRET peptide has been conveniently used in our Amplite Flurorimetric HIV Protease Assay Kit for high throughput screening of HIV-1 protease inhibitors and continuous quantification of HIV-1 protease activity. The sequence of this FRET peptide is derived from the native p17/p24 cleavage site on Prgag for HIV-1 protease. In the FRET peptide, the fluorescence of Tide Fluor™ 2 is quenched by Tide Quencher™ 2 until this peptide is cleaved into two separate fragments by HIV1 protease.

 

Tide Fluor™ Donor Dyes, Optimized to Maximize FRET Performance through Enhancing Donor Fluorescence Intensity


Although EDANS, FAM, TAMRA, ROX, Cy 3 and Cy5 have been widely used to develop a variety of FRET probes, there are still some limitations in the use of these dyes. For example, the weak absorption and environment-sensitive fluorescence of EDANS has severely limited its sensitivity for developing protease assays and nucleic acid detection probes. Compared to EDANS, fluorescein-based probes (such as FAM, HEX, JOE and TET) have stronger absorption and fluorescence. However the fluorescence of fluorescein-based probes is strongly dependent on pH. They only exhibit the strongest fluorescence at higher pH. This pH dependence makes the fluorescein-based fluorescent probes inconvenient for the assays that require low pH. In addition, most of fluorescein-based probes have quite low photostability, which limits their applications in fluorescence imaging. Among cyanine dyes, non-sulfonated Cy3 and Cy5 are widely used for developing a variety of nucleic acid probes, but they have quite low fluorescence quantum yield in aqueous media. The sulfonated Cy3 and Cy5 have improved fluorescence quantum yield than those of non-sulfonate cyanines. However, the sulfonated Cy3 and Cy5 are difficult to use in the synthesis of fluorescent oligonucleotides, and are quite cost-prohibitive.

Key Features of Tide Fluor™ Donors

  • Optimized to pair with Tide Quencher™ dark acceptors to maximize the FRET potentials
  • Stronger fluorescence intensity to enhance assay sensitivity
  • pH-insensitive and environment-insensitive fluorescence to simplify assays
  • Higher photostability to improve the quality of fluorescence imaging
  • A variety of reactive forms available for conjugations
 

Table 1. Tide Fluor™ Dyes for Developing FRET Probes

Tide Fluor™ Donor
Ex (nm)
Em (nm)
Features and Benefits
Ordering Information
Tide Fluor™ 1 (TF1)345442
  • Alternative to EDANS
  • Much stronger absorption
  • Much stronger fluorescence
  • Less environmental sensitivity
Cat# 2236 (TF1 azide, click chemistry)
Cat# 2237 (TF1 alkyne, click chemistry)
Cat# 2238 (TF1 acid)
Cat# 2239 (TF1 amine)
Cat# 2242 (TF1 maleimide, SH-reactive)
Cat# 2244 (TF1 SE, NH2-reactive)
Tide Fluor™ 2 (TF2)500527
  • Alternative to FAM, FITC and Alexa Fluor® 488
  • pH-insensitive fluorescence
  • Good photostability
Cat# 2245 (TF1 acid)
Cat# 2246 (TF2 amine)
Cat# 2247 (TF2 maleimide SH-reactive)
Cat# 2248 (TF2 SE, NH2-reactive)
Cat# 2252 (TF2 azide, click chemistry)
Cat# 2253 (TF2 alkyne, click chemistry)
Tide Fluor™ 3 (TF3)555584
  • Alternative to Cy3® and Alexa Fluor® 555
  • Strong fluorescence
  • Good photostability
Cat# 2254 (TF3 azide, click chemistry)
Cat# 2255 (TF3 alkyne, click chemistry)
Cat# 2268 (TF3 acid)
Cat# 2269 (TF3 amine)
Cat# 2270 (TF3 maleimide, SH-reactive)
Cat# 2271 (TF3 SE, NH2-reactive)
Tide Fluor™ 3WS (TF3WS)555565
  • Alternative to Cy3® and Alexa Fluor® 555
  • Strong fluorescence
  • Good photostability
Cat# 2345 (TF3WS acid)
Cat# 2346 (TF3WS SE, NH2-reactive)
Tide Fluor™ 4 (TF4)590618
  • Alternative to ROX, Texas Red® and Alexa Fluor® 594
  • Strong fluorescence
  • Good photostability
Cat# 2285 (TF4 acid)
Cat# 2286 (TF4 amine)
Cat# 2287 (TF4 maleimide, SH-reactive)
Cat# 2289 (TF4 SE, NH2-reactive)
Cat# 2300 (TF4 azide, click chemistry)
Cat# 2301 (TF4 alkyne, click chemistry)
Tide Fluor™ 5WS (TF5WS)649664
  • Alternative to Cy5® and Alexa Fluor® 647
  • Strong fluorescence
  • Good photostability
Cat# 2275 (TF5WS azide, click chemistry)
Cat# 2276 (TF5WS alkyne, click chemistry)
Cat# 2278 (TF5WS, acid)
Cat# 2279 (TF5WS amine)
Cat# 2280 (TF5WS maleimide, SH-reactive)
Cat# 2281 (TF5WS SE, NH2-reactive)
Tide Fluor™ 6WS (TF6WS)676695
  • Alternative to Cy5.5®, IRDye® 700 and Alexa Fluor® 680
  • Strong fluorescence
  • Good photostability
Cat# 2291 (TF6WS acid)
Cat# 2292 (TF6WS amine)
Cat# 2293 (TF6WS maleimide, SH-reactive)
Cat# 2294 (TF6WS SE, NH2-reactive)
Cat# 2302 (TF6WS azide, click chemistry)
Cat# 2303 (TF6WS alkyne, click chemistry)
Tide Fluor™ 7WS (TF7WS)749775
  • Alternative to Cy7® and Alexa Fluor® 750
  • Strong fluorescence
  • Good photostability
Cat# 2304 (TF7WS azide, click chemistry)
Cat# 2305 (TF7WS alkyne, click chemistry)
Cat# 2330 (TF7WS acid)
Cat# 2331 (TF7WS amine)
Cat# 2332 (TF7WS maleimide, SH-reactive)
Cat# 2333 (TF7WS SE, NH2-reactive)
Tide Fluor™ 8WS (TF8WS)775807
  • Alternative to IRDye® 800
  • Strong fluorescence
  • Good photostability
Cat# 2306 (TF8WS azide, click chemistry)
Cat# 2307 (TF8WS alkyne, click chemistry)
Cat# 2335 (TF8WS acid)
Cat# 2336 (TF8WS amine)
Cat# 2337 (TF8WS maleimide, SH-reactive)
Cat# 2338 (TF8WS SE, NH2-reactive)
  1. *Texas Red® is the trademark of Molecular Probes, Inc.

To address these limitations, we have developed Tide Fluor™ donor dyes that are optimized as building blocks for developing FRET oligonucleotides and peptides for a variety of biological applications. Our Tide Fluor™ dyes (such as TF1, TF2, TF3, TF4, TF5, TF6, TF7 and TF8) have stronger fluorescence and higher photostability than the typical fluorophores such as fluoresceins, rhodamines and cyanines as described below. Our TF2 has the essentially same excitation and emission wavelengths to those of carboxyfluoresceins (FAM), making them readily used for the biological applications that were done with fluoresceins, but have enhanced performance with our TF2 probes. Compared to FAM probes, TF2 has much stronger fluorescence at physiological conditions, and it is much more photostable. Compared to other fluorescent dyes alternative to fluoresceins and Cy dyes (such as Alexa Fluor™ and Cy3, Cy5 and Cy7), Tide Fluor™ dyes are much more cost-effective with comparable or even better performance for your desired biological applications.

 

Tide Quencher™ Acceptor Dyes, Optimized to Maximize FRET Performance through Enhancing Quenching Efficiency


Spectral Overlap
The spectral overlap of Tide Quencher™ 1 (TQ1, top) and Tide Quencher™ 2 (TQ2, bottom) with EDANS (top) and FAM (bottom).
Although DABCYL has been used to develop a variety of FRET applications, its low quenching efficiency of longer wavelength dyes (such as fluoresceins, rhodamines and cyanines) has limited its use in the development of sensitive fluorogenic FRET probes. Additionally, the absorption spectrum of DABCYL is environment-sensitive. AAT Bioquest has developed the robust Tide Quencher™ acceptor dyes for the development of longer wavelength FRET probes. These Tide Quencher™ dark FRET acceptors (such as TQ1, TQ2, TQ3, TQ4, TQ5, TQ6 and TQ7) are optimized to pair with our Tide Fluor™ dyes and the classic fluorophores (such as AMCA, EDANS, FAM, TAMRA, HEX, JOE, TET, ROX, Cy3, Cy5 and Cy7). Like our Tide Fluor™ donors dyes, our Tide Quencher™ acceptor dyes are much more cost-effective with comparable or even better performance for your desired biological applications than other similar products on the market.

Besides their broad applications in the development of Molecular Beacon probes, our Tide Quencher™ dyes have also been used to develop various protease substrates such as HIV protease (see above), MMPs and secretases. In some cases, it has demonstrated greatly improved enzyme performance. This may be partly due to the red-shifted absorption spectrum that overlaps better with the emission spectrum of fluoresceins, rhodamines and cyanines. Tide Quencher™ dyes are great choice for you to eliminate the limitations of classic quenchers. Tide Quencher ™ dyes are excellent dark quenchers that are individually optimized to pair with all of the popular fluorescent dyes such as fluoresceins and rhodamines. Our Tide Quencher™ series of nonfluorescent dyes cover the full visible spectrum with unusually high efficiency. TQ2 has absorption maximum perfectly matching the emission of FAM (See Figure 6) while TQ3 is proven to be the best quencher for TAMRA and Cy3. In summary, our Tide Quencher™ dyes have the following advantages:
  • Most Powerful: enable you to explore the FRET potentials that might be impossible with other quenchers.
  • Versatile Reactive Forms: convenient for self-constructing your desired FRET biomolecules.
  • A Complete Set of Dyes: perfectly match your desired fluorescent donors.
  • Enhanced Value: competitive price with the best performance.
 

Table 2. Tide Quencher™ Dyes for Developing FRET Probes

Dark FRET Acceptor
λmax (nm)
Features and Benefits
Ordering Information
Tide Quencher™ 1 (TQ1)490
  • Alternative to Dabcyl, QSY® 35 and BHQ®-0
  • Best paired with Tide Fluor™ 1 (TF1)
  • Excellent FRET efficiency with coumarins
Cat# 2188 (TQ1 azide, click chemistry)
Cat# 2189 (TQ1 alkyne, click chemistry)
Cat# 2190 (TQ1 acid)
Cat# 2192 (TQ1 amine)
Cat# 2193 (TQ1 CPG, OH-reactive)
Cat# 2196 (TQ1 maleimide, SH-reactive)
Cat# 2198 (TQ1 phosphoramidite, OH-reactive)
Cat# 2199 (TQ1 SE, NH2-reactive)
Tide Quencher™ 2 (TQ2)515
  • Alternative to BHQ®-1
  • Best paired with Tide Fluor™ 2 (TF2)
  • Better matched with FAM, FITC and Alexa Fluor® 488 than other commercial quenchers
Cat# 2211 (TQ2 azide, click chemistry)
Cat# 2212 (TQ2 alkyne, click chemistry)
Cat# 2200 (TQ2 acid)
Cat# 2202 (TQ2 amine)
Cat# 2203 & 2204 (TQ2 CPG, OH-reactive)
Cat# 2206 (TQ2 maleimide, SH-reactive)
Cat# 2208 (TQ2 phosphoramidite, OH-reactive)
Cat# 2210 (TQ2 SE, NH2-reactive)
Tide Quencher™ 2WS (TQ2WS)515
  • Alternative to BHQ®-1
  • Best paired with Tide Fluor™ 2 (TF2)
  • Better matched with FAM, FITC and Alexa Fluor® 488 than other commercial quenchers
Cat# 2050 (TQ2WS acid)
Cat# 2058 (TQ2WS SE, NH2-reactive)
Tide Quencher™ 3 (TQ3)570
  • Alternative to QSY® 7, QSY® 9 and BHQ®-2
  • Best paired with Tide Fluor™ 3 (TF3)
  • Excellent FRET efficiency with Cy3®, Alexa Fluor® 555 and TAMRA than other commercial quenchers
Cat# 2200 (TQ3 acid)
Cat# 2222 (TQ3 amine)
Cat# 2223 & 2224 (TQ3 CPG, OH-reactive)
Cat# 2226 (TQ3 maleimide, SH-reactive)
Cat# 2228 (TQ3 phosphoramidite, OH-reactive)
Cat# 2230 (TQ3 SE, NH2-reactive)
Cat# 2231 (TQ3 azide, click chemistry)
Cat# 2232 (TQ3 alkyne, click chemistry)
Tide Quencher™ 3WS (TQ3WS)578
  • Alternative to QSY® 7, QSY® 9 and BHQ®-2
  • Best paired with Tide Fluor™ 3 (TF3)
  • Excellent FRET efficiency with Cy3®, Alexa Fluor® 555 and TAMRA than other commercial quenchers
Cat# 2227 (TQ3WS acid)
Cat# 2229 (TQ3WS SE, NH2-reactive)
Tide Quencher™ 4 (TQ4)603
  • Strong absorption
  • Best paired with Tide Fluor™ 4 (TF4)
  • Better FRET efficiency with ROX, Texas Red® and Alexa Fluor® 594 than other commercial quenchers
Cat# 2062 & 2063 (TQ4 CPG, OH-reactive)
Tide Quencher™ 4WS (TQ4WS)~590
  • Strong absorption
  • Best paired with Tide Fluor™ 4 (TF4)
  • Better FRET efficiency with ROX, Texas Red® and Alexa Fluor® 594 than other commercial quenchers
Cat# 2060 (TQ4WS acid)
Cat# 2061 (TQ4WS amine)
Cat# 2064 (TQ4WS maleimide, SH-reactive)
Cat# 2067 (TQ4WS SE, NH2-reactive)
Cat# 2068 (TQ4WS azide, click chemistry)
Cat# 2069 (TQ4WS alkyne, click chemistry)
Tide Quencher™ 5 (TQ5)~670
  • Alternative to QSY® 21 and BHQ®-3
  • Best paired with Tide Fluor™ 5 (TF5)
  • Excellent FRET efficiency with Cy5®, DyLight® 649 and Alexa Fluor®647
Cat# 2077 & 2078 (TQ5 CPG, OH-reactive)
Tide Quencher™ 5WS (TQ5WS)~670
  • Alternative to QSY® 21 and BHQ®-3
  • Best paired with Tide Fluor™ 5 (TF5)
  • Excellent FRET efficiency with Cy5®, DyLight® 649 and Alexa Fluor®647
Cat# 2075 (TQ5WS acid)
Cat# 2076 (TQ5WS amine)
Cat# 2079 (TQ5WS maleimide, SH-reactive)
Cat# 2081 (TQ5WS SE, NH2-reactive)
Cat# 2082 (TQ5WS azide, click chemistry)
Cat# 2083 (TQ5WS alkyne, click chemistry)
Tide Quencher™ 6WS (TQ6WS)~700
  • Stronger absorption
  • Best paired with Tide Fluor™ 6 (TF6)
  • Better FRET efficiency with Cy5.5®, IRDye® 700 and Alexa Fluor® 680 than other commercial quenchers
Cat# 2090 (TQ6WS acid)
Cat# 2091 (TQ6WS amine)
Cat# 2094 (TQ6WS maleimide, SH-reactive)
Cat# 2096 (TQ6WS SE, NH2-reactive)
Cat# 2097 (TQ6WS azide, click chemistry)
Cat# 2098 (TQ5WS alkyne, click chemistry)
Tide Quencher™ 7WS (TQ7WS)~760
  • Stronger absorption
  • Best paired with Tide Fluor™ 7 (TF7)
  • Better FRET efficiency with Cy7® and Alexa Fluor® 750 than other commercial quenchers
Cat# 2105 (TQ7WS acid)
Cat# 2106 (TQ7WS amine)
Cat# 2109 (TQ7WS maleimide, SH-reactive)
Cat# 2111 (TQ7WS SE, NH2-reactive)
Cat# 2112 (TQ7WS azide, click chemistry)
Cat# 2113 (TQ7WS alkyne, click chemistry)
  1. *Texas Red® is the trademark of Molecular Probes, Inc.

 

Selection of FRET Donors and Acceptors


Probes incorporating fluorescent donor/non-fluorescent acceptor (e.g. Dabcyl) combinations have been developed primarily for the detection of proteolysis and nucleic acid hybridization. This principle has also been used to develop other FRET-based assays such as receptor/ligand interactions, distribution and transport of lipids, membrane potential sensing and cyclic AMP detection.

The donor and acceptor molecules can be the same or different. In most applications, they are different dyes. FRET can be detected either by the appearance of sensitized fluorescence of the acceptor or by the intensity ratio change of donor/acceptor (if the acceptor is fluorescent), or the fluorescence decrease of the donor. In the later case, acceptor can be either fluorescent or non-fluorescent. A number of Ro values for various D/A pairs have been published. Larger Ro values of FRET pairs give higher FRET efficiency. Fluorescein/tetramethylrhodamine pairs have been most widely used to develop FRET-based assays. EDANS/DABCYL pair has been intensively used to develop FRET-based nucleic acid probes and protease substrates. MCA (7-methoxycoumarin-4-acetic acid) and DNP (2, 4-dinitroaniline) are another pair of donor/acceptor for developing FRET-based fluorescent probes. Compared with the pair of EDANS/DABCYL, MCA/DNP pair usually has shorter and weaker wavelength of fluorescence signal. However, the pair often demonstrates better affinity or turnover rate due to their smaller sizes. DNP is also a good FRET acceptor paired with tryptophan (Trp), 2-aminobenzoic acid (Abz) or Abz derivatives such as Abz(N-Me).
 

Table 3. Common FRET donor and acceptor pairs and their R0 values.

Donor
Acceptor
R0
B-PhycoerythrinCy579
DansylFluorescein33-41
EDANSDABCYL33
FluoresceinFluorescein44
FluoresceinTetramethylrhodamine49-56
IAEDANS*5-IAF (5-Iodoacetamidofluorescein)49
IAEDANS*FITC49
NaphthaleneDansyl22
PyreneCoumarin39
TryptophanDansyl21-24
TryptophanPyrene28
TryptophanIAEDANS*22
R0(Å)Tetramethylrhodamine49-56
  1. *The value may change under different conditions; **IAEDANS = 5-((Iodoacetyl)amino)naphthalene-sulfonic acid.

Table 4 summarizes our Tide Quencher™ FRET building blocks designed for you to develop FRET probes for the demanding applications. Based on our in-house research and experience, it is recommended to use the FRET pairs marked in green. The pairs marked in cyan color are OK to use, but less efficient than the ones marked in green. We have proved that these recommended FRET pairs have demonstrated high sensitivity and low background in our protease and nucleic acid detection assays. There are also other factors that you need to consider besides the FRET efficiency, such as pH, multiplexing and buffer interference, etc.

Table 4

Table 4. Tide Quencher FRET Building Blocks

 

Ordering Information


 

Table 4. Ordering Info For Tide Quencher Products

Cat#
Product Name
Unit Size
2170Tide Quencher™ 2WS acid [TQ2WS acid]25 mg
2178Tide Quencher™ 2WS succinimidyl ester [TQ2WS, SE]5 mg
2179Tide Quencher™ 2WS maleimide [TQ2WS maleimide]1 mg
2060Tide Quencher™ 4WS acid [TQ4WS acid]5 mg
2061Tide Quencher™ 4WS amine [TQ4WS amine]1 mg
2062Tide Quencher™ 4 CPG [TQ4 CPG] *500 Å*100 mg
2063Tide Quencher™ 4 CPG [TQ4 CPG] *1000 Å*100 mg
2064Tide Quencher™ 4WS maleimide [TQ4WS maleimide]1 mg
2067Tide Quencher™ 4WS succinimidyl ester [TQ4WS SE]1 mg
2068Tide Quencher™ 4WS azide [TQ4WS azide]1 mg
2069Tide Quencher™ 4WS alkyne [TQ4WS alkyne]1 mg
2075Tide Quencher™ 5WS acid [TQ5WS acid]5 mg
2076Tide Quencher™ 5WS amine [TQ5WS amine]1 mg
2077Tide Quencher™ 5 CPG [TQ5 CPG] *500 Å*100 mg
2078Tide Quencher™ 5 CPG [TQ5 CPG] *1000 Å*100 mg
2079Tide Quencher™ 5WS maleimide [TQ5WS maleimide]1 mg
2081Tide Quencher™ 5WS succinimidyl ester [TQ5WS SE]1 mg
2082Tide Quencher™ 5WS azide [TQ5WS azide]1 mg
2083Tide Quencher™ 5WS alkyne [TQ5WS alkyne]1 mg
2090Tide Quencher™ 6WS acid [TQ6WS acid]5 mg
2091Tide Quencher™ 6WS amine [TQ6WS amine]1 mg
2094Tide Quencher™ 6WS maleimide [TQ6WS maleimide]1 mg
2096Tide Quencher™ 6WS succinimidyl ester [TQ6WS SE]1 mg
2097Tide Quencher™ 6WS azide [TQ6WS azide]1 mg
2098Tide Quencher™ 6WS alkyne [TQ6WS alkyne]1 mg
2105Tide Quencher™ 7WS acid [TQ7WS acid]5 mg
2106Tide Quencher™ 7WS amine [TQ7WS amine]1 mg
2109Tide Quencher™ 7WS maleimide [TQ7WS maleimide]1 mg
2111Tide Quencher™ 7WS succinimidyl ester [TQ7WS SE]1 mg
2112Tide Quencher™ 7WS azide [TQ7WS azide]1 mg
2113Tide Quencher™ 7WS alkyne [TQ7WS alkyne]1 mg
2188Tide Quencher™ 1 azide [TQ1 azide]5 mg
2189Tide Quencher™ 1 alkyne [TQ1 alkyne]5 mg
2190Tide Quencher™ 1 acid [TQ1 acid]100 mg
2192Tide Quencher™ 1 amine [TQ1 amine]5 mg
2193Tide Quencher™ 1 CPG [TQ1 CPG] *500 Å*100 mg
2194Tide Quencher™ 1 CPG [TQ1 CPG] *1000 Å*100 mg
2196Tide Quencher™ 1 maleimide [TQ1 maleimide]5 mg
2198Tide Quencher™ 1 phosphoramidite [TQ1 phosphoramidite]100 umoles
2199Tide Quencher™ 1 succinimidyl ester [TQ1 SE]25 mg
2200Tide Quencher™ 2 acid [TQ2 acid]100 mg
2202Tide Quencher™ 2 amine [TQ2 amine]5 mg
2203Tide Quencher™ 2 CPG [TQ2 CPG] *500 Å*100 mg
2204Tide Quencher™ 2 CPG [TQ2 CPG] *1000 Å*100 mg
2208Tide Quencher™ 2 phosphoramidite [TQ2 phosphoramidite]100 umoles
2210Tide Quencher™ 2 succinimidyl ester [TQ2 SE]25 mg
2211Tide Quencher™ 2 azide [TQ2 azide]5 mg
2212Tide Quencher™ 2 alkyne [TQ2 alkyne]5 mg
2213Tide Quencher™ 2WS alkyne [TQ2WS alkyne]1 mg
2214Tide Quencher™ 2WS alkyne [TQ2WS alkyne]5 mg
2220Tide Quencher™ 3 acid [TQ3 acid]100 mg
2222Tide Quencher™ 3 amine [TQ3 amine]5 mg
2223Tide Quencher™ 3 CPG [TQ3 CPG] *500 Å*100 mg
2224Tide Quencher™ 3 CPG [TQ3 CPG] *1000 Å*100 mg
2226Tide Quencher™ 3 maleimide [TQ3 maleimide]5 mg
2227Tide Quencher™ 3WS acid [TQ3WS acid]5 mg
2228Tide Quencher™ 3 phosphoramidite [TQ3 phosphoramidite]100 umoles
2229Tide Quencher™ 3WS succinimidyl ester [TQ3WS SE]1 mg
2230Tide Quencher™ 3 succinimidyl ester [TQ3 SE]25 mg
2231Tide Quencher™ 3 azide [TQ3 azide]5 mg
2232Tide Quencher™ 3 alkyne [TQ3 alkyne]5 mg


Table 5. Tide Fluor™ dyes for labeling peptides, oligonucleotides and other biomolecules.

Product Name
Unit Size
Cat#
FMOC-Asp(TF3)-OH100 mg5007
FMOC-Glu(TF3)-OH100 mg5016
FMOC-Lys(TF3)-OH100 mg5051
Tide Fluor™ 1 acid [TF1 acid] *Superior replacement for EDANS*100 mg2238
Tide Fluor™ 1 alkyne [TF1 alkyne]5 mg2237
Tide Fluor™ 1 amine [TF1 amine] *Superior replacement for EDANS*5 mg2239
Tide Fluor™ 1 azide [TF1 azide]5 mg2236
Tide Fluor™ 1 CPG [TF1 CPG] *1000 Å* *Superior replacement for EDANS*100 mg2241
Tide Fluor™ 1 CPG [TF1 CPG] *500 Å* *Superior replacement for EDANS*100 mg2240
Tide Fluor™ 1 maleimide [TF1 maleimide]5 mg2242
Tide Fluor™ 1 succinimidyl ester [TF1 SE]*Superior replacement for EDANS*5 mg2244
Tide Fluor™ 2 acid [TF2 acid] *Superior replacement for fluorescein*25 mg2245
Tide Fluor™ 2 alkyne [TF2 alkyne]1 mg2253
Tide Fluor™ 2 amine [TF2 amine] *Superior replacement for fluorescein*1 mg2246
Tide Fluor™ 2 azide [TF2 azide]1 mg2252
Tide Fluor™ 2 maleimide [TF2 maleimide] *Superior replacement for fluorescein*1 mg2247
Tide Fluor™ 2, succinimidyl ester [TF2 SE]*Superior replacement for fluorescein*5 mg2248
Tide Fluor™ 2WS acid [TF2WS acid] *Superior replacement for FITC*10 mg2348
Tide Fluor™ 2WS amine [TF2WS amine] *Superior replacement for FITC*1 mg2351
Tide Fluor™ 2WS maleimide [TF2WS Maleimide] *Superior replacement for FITC*1 mg2350
Tide Fluor™ 2WS succinimidyl ester [TF2WS SE] *Superior replacement for FITC*5 mg2349
Tide Fluor™ 3 acid [TF3 acid] *Superior replacement for Cy3*25 mg2268
Tide Fluor™ 3 alkyne [TF3 alkyne]1 mg2255
Tide Fluor™ 3 amine [TF3 amine] *Superior replacement for Cy3*1 mg2269
Tide Fluor™ 3 azide [TF3 azide]1 mg2254
Tide Fluor™ 3 maleimide [TF3 maleimide] *Superior replacement for Cy3*1 mg2270
Tide Fluor™ 3 phosphoramidite [TF3 CEP] *Superior replacement to Cy3  phosphoramidite*100 µmoles2274
Tide Fluor™ 3 succinimidyl ester [TF3 SE]*Superior replacement for Cy3*5 mg2271
Tide Fluor™ 3WS acid [TF3WS acid] *Superior replacement for Cy3*10 mg2345
Tide Fluor™ 3WS amine [TF3WS amine] *Superior replacement for Cy3*1 mg2347
Tide Fluor™ 3WS maleimide [TF3WS maleimide] *Superior replacement for Cy3*1 mg2344
Tide Fluor™ 3WS succinimidyl ester [TF3WS SE] *Superior replacement for Cy3*5 mg2346
Tide Fluor™ 4 acid [TF4 acid] *Superior replacement for ROX and Texas Red*10 mg2285
Tide Fluor™ 4 alkyne [TF4 alkyne]1 mg2301
Tide Fluor™ 4 amine [TF4 amine] *Superior replacement for ROX and Texas Red*1 mg2286
Tide Fluor™ 4 azide [TF4 azide]1 mg2300
Tide Fluor™ 4 maleimide [TF4 maleimide] *Superior replacement for ROX and Texas Red*1 mg2287
Tide Fluor™ 4, succinimidyl ester [TF4 SE]*Superior replacement for ROX and Texas Red*5 mg2289
Tide Fluor™ 5WS acid [TF5WS acid] *Superior replacement for Cy5*10 mg2278
Tide Fluor™ 5WS alkyne [TF5WS alkyne]1 mg2276
Tide Fluor™ 5WS amine [TF5WS amine] *Superior replacement for Cy5*1 mg2279
Tide Fluor™ 5WS azide [TF5WS azide]1 mg2275
Tide Fluor™ 5WS maleimide [TF5WS maleimide] *Superior replacement for Cy5*1 mg2280
Tide Fluor™ 5WS succinimidyl ester [TF5WS SE]*Superior replacement for Cy5*5 mg2281
Tide Fluor™ 6WS acid [TF6WS acid] *Superior replacement for Cy5.5*10 mg2291
Tide Fluor™ 6WS alkyne [TF6WS alkyne]1 mg2303
Tide Fluor™ 6WS amine [TF6WS amine] *Superior replacement for Cy5.5*1 mg2292
Tide Fluor™ 6WS azide [TF6WS azide]1 mg2302
Tide Fluor™ 6WS maleimide [TF6WS maleimide] *Superior replacement for Cy5.5*1 mg2293
Tide Fluor™ 6WS succinimidyl ester [TF6WS SE]*Superior replacement for Cy5.5*1 mg2294
Tide Fluor™ 7WS acid [TF7WS acid] *Superior replacement for Cy7*10 mg2330
Tide Fluor™ 7WS alkyne [TF7WS alkyne]1 mg2305
Tide Fluor™ 7WS amine [TF7WS amine] *Superior replacement for Cy5.5*1 mg2331
Tide Fluor™ 7WS azide [TF7WS azide]1 mg2304
Tide Fluor™ 7WS maleimide [TF7WS maleimide] *Superior replacement for Cy5.5*1 mg2332
Tide Fluor™ 7WS, succinimidyl ester [TF7WS SE]*Superior replacement for Cy7*1 mg2333
Tide Fluor™ 8WS acid [TF8WS acid] *Near Infrared Emission*10 mg2335
Tide Fluor™ 8WS alkyne [TF8WS alkyne]1 mg2307
Tide Fluor™ 8WS amine [TF8WS amine] *Superior replacement for Cy5.5*1 mg2336
Tide Fluor™ 8WS azide [TF8WS azide]1 mg2306
Tide Fluor™ 8WS maleimide [TF8WS maleimide] *Superior replacement for Cy5.5*1 mg2337
Tide Fluor™ 8WS, succinimidyl ester [TF8WS SE]*Near Infrared Emission*1 mg2338

 

References


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Original created on January 2, 2020, last updated on November 8, 2022
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