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20 Chapter 1 — Fluorophores and Their Amine-Reactive Derivatives www.probes.com

1.3 Alexa Fluor Dyes: Simply the BestMolecular Probes’ Alexa Fluor dyes (see The Alexa Fluor Dye Series — Peak Perfor-

mance Across the Visible Spectrum) set new standards for fluorophores and the biocon-jugates prepared from them. The absorption spectra (Figure 1.14, Figure 1.21, Figure1.30) of these spectrally distinct dyes — Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and AlexaFluor 750 dyes — cover the entire spectrum and match the principal output wavelengthsof common excitation sources.1

With spectra almost identical to those of fluorescein (Figure 1.8), but with far greaterfluorescence in its conjugates and significantly better photostability, the Alexa Fluor 488dye is indisputably the best green-fluorescent reactive dye available. Spectra of the AlexaFluor 555 dye are an almost perfect match to those of the Cy3 dye (Figure 1.15), butbioconjugates of the Alexa Fluor 555 dye are more fluorescent (Figure 1.25) and morephotostable (Figure 1.17) than those of the Cy3 dye. Similarly, spectra of the Alexa Fluor647 conjugates substantially match those of the Cy5 dye (Figure 1.22) and the AlexaFluor 680 and Alexa Fluor 750 dyes match the spectral properties of the Cy5.5 and Cy7dyes, respectively (Figure 1.23, Figure 1.24); however, the Alexa Fluor dyes usuallyprovide superior performance, particularly in their protein and nucleic acid conjugates.Tandem conjugates of the Alexa Fluor dyes with R-phycoerythrin and allophycocyanin(Section 6.4) further expand the utility of the Alexa Fluor dyes in multicolor applications(Figure 6.31, Figure 6.34).

Zenon One Mouse IgG1 Labeling Kits are available for all of our Alexa Fluor dyes(Table 1.1, Table 7.1). Our exclusive Zenon reagents (Section 7.2) make it possible torapidly and quantitatively form complexes of any whole mouse IgG1 antibody (Figure7.32).

The Alexa Fluor series of dyes shares several significant attributes, including:

• High absorbance at wavelengths of maximal output of common excitation sources• Bright and unusually photostable fluorescence of their bioconjugates• Good water solubility of the reactive dyes for ease of conjugation and resistance of the

conjugates to precipitation and aggregation• Insensitivity of their spectra to pH over a broad range• Well-differentiated spectra, providing many options for multicolor detection and

fluorescence resonance energy transfer (see Fluorescence Resonance Energy Transfer(FRET))

Figure 1.8 Absorption and fluorescence emissionspectra of fluorescein goat anti–mouse IgG anti-body (F-2761, (blue)) and Alexa Fluor 488 goatanti–mouse IgG antibody (A-11001, (red)). The flu-orescence intensity of the Alexa Fluor 488 conju-gate was significantly higher than that of the fluo-rescein conjugate. The data are normalized toshow the spectral similarity.

Figure 1.9 Bovine pulmonary artery endothelialcells (BPAEC) were labeled with fluorescein phalloi-din (left panels, F-432) or Alexa Fluor 488 phalloidin(right panels, A-12379), which labels filamentousactin, and mounted in PBS. The cells were placedunder constant illumination on the microscope withan FITC filter set using a 60× objective. Images wereacquired at one-second intervals for 30 seconds.Under these illumination conditions, fluoresceinphotobleached to about 20% of its initial value in 30seconds; the fluorescence of Alexa Fluor 488 phal-loidin stayed at the initial value under the same illu-mination conditions.

Figure 1.10 Comparison of the photobleaching rates of the Alexa Fluor 488 and Alexa Fluor 546 dyes andthe well-known fluorescein and Cy3 fluorophores. The cytoskeleton of bovine pulmonary artery endothelialcells (BPAEC) was labeled with (top series) Alexa Fluor 488 phalloidin (A-12379) and mouse monoclonalanti–α-tubulin antibody (A-11126) in combination with Alexa Fluor 546 goat anti–mouse IgG antibody(A-11003) or (bottom series) fluorescein phalloidin (F-432) and the anti–α-tubulin antibody in combinationwith a commercially available Cy3 goat anti–mouse IgG antibody. The pseudocolored images were taken at30-second intervals (0, 30, 90, and 210 seconds of exposure). The images were acquired with bandpass fil-ter sets appropriate for fluorescein and rhodamine.

The number following our Alexa Fluorregistered trademark represents theapproximate absorption maximum ofthe dye. In most cases these wave-lengths correspond to intense spectrallines of common laser, laser diode orbroadband excitation sources.

21Section 1.3

TECHNICAL NOTE

The Alexa Fluor Dye Series — Peak Performance Across the Visible SpectrumThe Alexa Fluor dyes — a series of new, superior fluorescent

dyes that span the visible spectrum — represent a major break-through in the development of fluorescent labeling reagents, espe-cially when combined with our multipurpose Zenon technology(Section 7.2). These dyes, without exception, produce the best andbrightest conjugates we have ever tested. Benefits of the AlexaFluor dyes and their conjugates include:

• Brightness — Alexa Fluor conjugates exhibit more intensefluorescence than other spectrally similar conjugates.

• Photostability — Alexa Fluor conjugates are more photostablethan most other fluorescent conjugates, allowing more time forimage capture (Figure 1.10).

• Instrument compatibility — Absorption spectra of the AlexaFluor conjugates are matched to the principal output wave-lengths of common excitation sources.

• Color selection — Alexa Fluor conjugates are available in sever-al distinct fluorescent colors, ranging from blue to red.

• pH insensitivity — Alexa Fluor dyes remain highly fluorescentover a broad pH range.

• Water solubility — Alexa Fluor reactive dyes have good watersolubility, so protein conjugations can be performed withoutorganic solvents, and the conjugates are relatively resistant toprecipitation during storage.

Alexa Fluor 350 Dye — Bright Blue and UV Light–ExcitableThe blue-fluorescent Alexa Fluor 350 dye produces conjugates

that are typically greater than 50% more fluorescent than conjugatesprepared from AMCA (Figure 7.28). Furthermore, because AlexaFluor 350 conjugates have slightly shorter-wavelength emissionmaxima than AMCA conjugates (442 nm versus 448 nm), the fluo-rescence of Alexa Fluor 350 conjugates is better separated from thatof commonly used green fluorophores (Figure 1.89, Figure 1.90).

Alexa Fluor 430 Dye — Absorption at 430 nmwith a High Stokes Shift

Few reactive dyes that absorb between 400 nm and 450 nmhave appreciable fluorescence beyond 500 nm in aqueous solution.Our Alexa Fluor 430 dye fills this spectral gap. Excitation near itsabsorption maximum at ~430 nm is accompanied by strong emis-sion near 540 nm (Figure 7.4, Figure 7.78).

Alexa Fluor 488 Dye — The Best Green FluorophoreProtein conjugates prepared with the Alexa Fluor 488 dye are far

superior to conjugates of fluorescein, and are indeed much betterthan conjugates of any other green fluorophore that we have tested,including those of the Cy2 dye (Figure 1.13). Not only are AlexaFluor 488 conjugates significantly brighter than fluorescein conju-gates (Figure 1.12), they are much more photostable (Figure 1.10,Figure 1.48, Figure 7.2). Also, fluorescence of the Alexa Fluor 488fluorophore is independent of pH from 4 to 10. This pH insensitivityis a major improvement over fluorescein, which emits fluorescencethat is significantly affected by pH (Figure 1.11, Figure 7.5, Figure7.47).

Alexa Fluor 532 Dye — The Optimal Dye for532 nm Excitation Sources

With excitation and emission spectra intermediate betweenthose of the green-fluorescent Alexa Fluor 488 dye and orange-fluorescent Alexa Fluor 546 dye (Figure 7.6), the Alexa Fluor 532dye and its conjugates are ideal for use with 532 nm excitationsources, including the frequency-doubled Nd:YAG laser (Figure11.18). The Alexa Fluor 532 dye is a preferred reporter dye fordetection of microsphere arrays on beads prepared by the LuminexLabMAP technology (www.luminexcorp.com/aroundthesphere/July_Aug2000/assayinfo.htm).

Alexa Fluor 546 Dye — A More Fluorescent Alternativeto Cy3 and Tetramethylrhodamine

Conjugates prepared with the Alexa Fluor 546 dye are perfect forapplications that require fluorescent probes that emit in the orangeregion of the spectrum. These intensely fluorescent conjugatesoutperform conjugates of tetramethylrhodamine (TRITC andTAMRA) and Cy3 (Figure 1.20, Figure 12.28) and are readily excitedby the strong 546 nm emission of mercury-arc lamps (Figure 6.8,Figure 7.7).

Alexa Fluor 555 Dye — A Superior Alternative tothe Cy3 Dye

Spectra of the Alexa Fluor 555 conjugates virtually match thoseof the Cy3 dye (Figure 1.15, Figure 7.8), resulting in an optimalmatch to filters designed for that dye. However, total fluorescenceof Alexa Fluor 555 conjugates is higher (Figure 1.19, Figure 1.25).The Alexa Fluor 555 dye is also more photostable (Figure 1.17),providing researchers with additional time for image capture.

Alexa Fluor 568 Dye — Perfect for 568 nmExcitation Sources

The red-orange–fluorescent Alexa Fluor 568 dye is optimallyexcited by the 568 nm spectral line of the Ar–Kr mixed-gas laserused in many confocal laser-scanning microscopes. Alexa Fluor568 conjugates are considerably brighter than LissamineRhodamine B conjugates or even Rhodamine Red-X conjugates,which have similar excitation and emission maxima (Figure 1.16,Figure 7.9).

Alexa Fluor 594 Dye — A Superior Alternative tothe Texas Red Dye

Conjugates prepared with the Alexa Fluor 594 dye emit in thered region of the spectrum (Figure 7.10), making them particularlyuseful for multilabeling experiments in combination with green-fluorescent probes. Alexa Fluor 594 conjugates are much morefluorescent than are Texas Red conjugates (Figure 1.18, Figure 7.77).

Alexa Fluor 633 Dye — The Optimal Dye for the 633 nmHe–Ne Laser Line

Far red-fluorescent dyes are among the most sought-after labelsfor fluorescence imaging because their spectra are well beyond the

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range of most sample autofluorescence. The growing popularity ofthe 633 nm spectral line of the He–Ne laser and the 635 nm spec-tral line of red diode lasers prompted us to create compatible dyes.Alexa Fluor 633 conjugates are bright and photostable, with a peakabsorbance centered at 632 nm and a peak emission centered at650 nm (Figure 7.11).

Alexa Fluor 647 Dye — A Superior Alternative tothe Cy5 Dye

Spectra of the Alexa Fluor 647 conjugates virtually match thoseof the Cy5 dye (Figure 1.22), resulting in an optimal match tooptical filters designed for that dye. However, total fluorescence ofthe secondary antibody conjugates of the Alexa Fluor 647 dye issignificantly higher than that of Cy5 conjugates supplied by othercompanies (Figure 1.26, Figure 1.27, Figure 1.28). Also, unlike theCy5 dye, the Alexa Fluor 647 dye has very little change in absor-bance or fluorescence spectra when conjugated to most proteins,oligonucleotides and nucleic acids (Figure 1.29), thus yieldinggreater total fluorescence at the same degree of substitution.

Alexa Fluor 660 Dye — An Optimal Dye for the647 nm Krypton-Ion Laser Line

The Alexa Fluor 660 dye is optimally excited with the 647 nmspectral line of the krypton-ion laser and well excited by the633 nm spectral line of the He–Ne laser. Protein conjugates of theAlexa Fluor 660 dye produce bright far-red–fluorescence emission,with a peak at 690 nm. The wide separation of its emission fromthat of other fluorophores allows use of the Alexa Fluor 660 dyewith other fluorescent labels, including the Alexa Fluor 546 and Cy3dyes and phycoerythrin conjugates (Figure 7.13). The Alexa Fluor

continued from previous page

660 dye is the dye of choice as a “second label” with allophycocya-nin (APC) conjugates in flow cytometry applications.

Alexa Fluor 680 Dye — An Alternative to the Cy5.5 DyeWith a peak excitation at 679 nm and maximum emission at

702 nm, the Alexa Fluor 680 dye is spectrally similar to the Cy5.5dye (Figure 1.23). Fluorescence emission of the Alexa Fluor 680dye is well separated from that of other commonly used red fluoro-phores, such as the tetramethylrhodamine, Texas Red, R-phyco-erythrin, Alexa Fluor 594 and Alexa Fluor 647 dyes, making it idealfor three- and four-color labeling (Figure 7.14).

Alexa Fluor 700 Dye — The Optimal Dye forFar-Red Diode Lasers

With an absorption maximum at 696 nm, the Alexa Fluor 700dye can be excited with a xenon-arc lamp, far-red diode lasers ordye-pumped lasers operating in the 675–700 nm range. The AlexaFluor 700 dye provides infrared fluorescence emission, with a peakat 719 nm (Figure 7.15).

Alexa Fluor 750 Dye — Our Longest-WavelengthAlexa Fluor Dye

Spectrally similar to the Cy7 dye (Figure 1.24), the Alexa Fluor750 dye is the longest-wavelength Alexa Fluor dye currently avail-able. Its fluorescence emission maximum at 779 nm is well sepa-rated from commonly used far-red fluorophores such as Alexa Fluor647, Alexa Fluor 660 or allophycocyanin (APC), facilitating multicol-or analysis. With a peak excitation at ~752 nm, conjugates of theAlexa Fluor 700 dye are well excited by a xenon-arc lamp or dye-pumped lasers operating in the 720–750 nm range (Figure 7.16).

Figure 1.11 Comparison of pH-dependent fluores-cence of the Oregon Green 488 (● ), carboxy-fluorescein ( ) and Alexa Fluor 488 ( ) fluoro-phores. Fluorescence intensities were measuredfor equal concentrations of the three dyes usingexcitation/emission at 490/520 nm.

• Extremely high FRET efficiency, with our calculated Ro values of up to 84 Å betweenpairs of Alexa Fluor dyes (Table 1.3) and up to 77 Å between Alexa Fluor dyes andsome nonfluorescent quenchers (Table 1.8)

Features of the Alexa Fluor Dyes

Alexa Fluor 488 DyeBased on our testing, publications 1–4 and results reported by customers (see Customer

Testimonials for the Alexa Fluor Dyes), the Alexa Fluor 488 dye is by far the best fluo-rescein (FITC or FAM) substitute available for most applications. It is probably the bestdye available for single-molecule detection of bioconjugates, for fluorescence correlationspectroscopy (FCS, see Fluorescence Correlation Spectroscopy (FCS)) and for fluores-cence polarization (FP, see Section 1.4) measurements. This green-fluorescent dye ex-hibits several unique features:

• Fluorescence spectra almost identical to those of fluorescein, with excitation/emissionmaxima of 491/515 nm (Figure 1.8) and a fluorescence lifetime of ~4.1 nanoseconds

• Strong absorption, with an extinction coefficient greater than 65,000 cm-1M-1

• Much more photostable than fluorescein (Figure 1.9, Figure 1.10), allowing more timefor observation and image capture

• pH-insensitive fluorescence between pH 4 and 10 (Figure 1.11)• Water soluble, with no organic co-solvents required in labeling reactions, suggesting

that the succinimidyl ester of Alexa Fluor 488 carboxylic acid (A-20000, A-20100) may

23Section 1.3

Table 1.3 R0 values for Alexa Fluor dyes.*

AcceptorDonor

AlexaFluor 488

AlexaFluor 546

AlexaFluor 555

AlexaFluor 568

AlexaFluor 594

AlexaFluor 647

Alexa Fluor350

50

Alexa Fluor488

NA 64 70 62 60 56

Alexa Fluor546

NA 70 71 74

Alexa Fluor555

NA 47 51

Alexa Fluor568

NA 82

Alexa Fluor594

NA 85

Alexa Fluor647

NA

* R0 values in angstroms (Å) represent the distance at which fluorescence resonance energy transfer from thedonor dye to the acceptor dye is 50% efficient. Values were calculated from spectroscopic data as outlined(see Fluorescence Resonance Energy Transfer (FRET) in Section 1.3). NA = Not applicable.

Figure 1.12 Comparison of the relative fluores-cence of goat anti–mouse IgG conjugates preparedfrom the Alexa Fluor 488 dye and from fluoresceinisothiocyanate (FITC). Conjugate fluorescence isdetermined by measuring the fluorescence quan-tum yield of the conjugated dye relative to that of areference dye and multiplying by the dye:proteinlabeling ratio.

Figure 1.13 Brightness comparison of MolecularProbes’ Alexa Fluor 488 goat anti–mouse IgG anti-body with Cy2 goat anti–mouse IgG antibody fromJackson ImmunoResearch. Human blood wasblocked with normal goat serum and incubatedwith an anti-CD3 mouse monoclonal antibody;cells were washed, resuspended and incubatedwith either Alexa Fluor 488 or Cy2 goat anti–mouseIgG antibody at equal concentration. Red bloodcells were lysed, and the samples were analyzedwith a flow cytometer equipped with a 488 nmargon-ion laser and a 525 ± 10 nm bandpass emis-sion filter.

be the ideal reagent for labeling amines of exposed cell-surface proteins of live cells• Superior fluorescence output per protein conjugate, surpassing that of any other spec-

trally similar fluorophore-labeled protein, including fluorescein conjugates (Figure1.12) and Cy2 conjugates of antibodies (Figure 1.13)

Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568 andAlexa Fluor 594 Dyes

These yellow- to orange- to red-fluorescent dyes (Figure 1.14) provide strong visiblefluorescence that contrasts well with the green fluorescence of the Alexa Fluor 488 dye;consequently, they are frequently used in combination with green-fluorescent dyes. Fiveof our Alexa Fluor dyes have been utilized for simultaneous seven-color fluorescenceimaging in tissue samples.5 The Alexa Fluor 532 dye (Figure 7.6) is readily excited bythe frequency-doubled output of the Nd:YAG laser. Both the Alexa Fluor 546 and AlexaFluor 555 dyes have spectra that are similar to tetramethylrhodamine and the Cy3 dye;the spectra of the Alexa Fluor 555 dye are an almost exact match to those of the Cy3 dye(Figure 1.15). The Alexa Fluor 568 (Figure 1.16) and Alexa Fluor 594 dyes have absorp-tion and fluorescence emission maxima similar to the Lissamine rhodamine B and TexasRed dyes, respectively. However, these orange- to red-fluorescent Alexa Fluor dyesexhibit several important distinguishing features:

• Excitation/emission maxima of ~553/569 nm for the Alexa Fluor 546 dye (Figure7.7), ~555/565 nm for the Alexa Fluor 555 dye (Figure 7.8), ~573/596 nm for theAlexa Fluor 568 dye (Figure 7.9) and ~585/610 nm for the Alexa Fluor 594 dye (Fig-ure 7.10), with fluorescence lifetimes for the Alexa Fluor 546, Alexa Fluor 568 andAlexa Fluor 594 dyes of approximately 4.0, 3.6 and 3.9 nanoseconds, respectively

• Strong absorption, with extinction coefficients greater than 80,000 cm-1M-1 for theAlexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 594 dyes andgreater than 150,000 cm-1M-1 for the Alexa Fluor 555 dye

• More photostable than other spectrally similar dyes, allowing more time for observa-tion and image capture (Figure 1.17)

• pH-insensitive fluorescence over a broad range• Water soluble, therefore permitting labeling reactions to be performed without organic

solvents• Superior fluorescence output per protein or nucleic acid conjugate, surpassing that of

any other spectrally similar fluorophore-labeled protein (Figure 1.18), including Cy3dye–labeled proteins (Figure 1.19, Figure 1.20)

Our experimental results indicate thatthe Alexa Fluor 488, Alexa Fluor 555,Alexa Fluor 647, Alexa Fluor 680 andAlexa Fluor 750 dyes generally providesuperior performance to the spectrallysimilar Cy2, Cy3, Cy5, Cy5.5 and Cy7dyes, respectively, particularly in theirprotein conjugates.


Isomeric mixtures of the amine-reactive monosuccinimidyl esters of the Alexa Fluor546, Alexa Fluor 568 and Alexa Fluor 594 dyes and the isomer-free monosuccinimidylester of the Alexa Fluor 555 dye are available as separate reagents in either a 1 mg or5 mg unit size or as components of several labeling kits (Table 1.1). The contents andutility of these protein and nucleic acid labeling kits are discussed in detail in Section 1.2.

Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660,Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 Dyes

A long-term goal at Molecular Probes has been to develop superior dyes that can beexcited by long-wavelength excitation sources, including the red He–Ne laser (at 633 nm),krypton-ion laser (at 647 nm) and laser diodes. It has particularly been a challenge toprepare reactive dyes whose fluorescence is not significantly quenched on conjugation.The Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor700 and Alexa Fluor 750 dyes (Figure 1.21) meet our goals in several ways:

• An excellent spectral match to common long-wavelength excitation sources, with veryhigh extinction coefficients — typically >165,000 cm-1M-1 but up to >230,000 cm-1M-1

for the Alexa Fluor 750 dye• Spectra of the Alexa Fluor 647, Alexa Fluor 680 and Alexa Fluor 750 conjugates that

virtually match those of the Cy5 dye (Figure 1.22), Cy5.5 dye (Figure 1.23) and Cy7dye (Figure 1.24), respectively, resulting in an optimal match to optical filters de-signed for these dyes (Table 24.6)

• Unusually low fluorescence quenching upon conjugation to proteins, even at relativelyhigh degrees of substitution (Figure 1.12, Figure 1.25, Figure 1.26), resulting in pro-tein conjugates that are typically at least three to four times brighter than those ofCy5, Cy5.5, Cy7 and similar dyes 6 but that are, in some cases, as much as 40-foldbrighter at equal antibody concentrations (Figure 1.23, Figure 1.26, Figure 1.27,Figure 1.28).

• Fluorescence of the nucleotide, oligonucleotide and nucleic acid conjugates of theAlexa Fluor 647 dye that usually exceeds that of the Cy5 dye conjugates (Section 8.2,Section 8.5)

• Unlike the Cy5 dye, very little change in absorbance or fluorescence spectra whenconjugated to most proteins, oligonucleotides and nucleic acids (Figure 1.29), thusyielding significantly greater total fluorescence at the same degrees of substitution(Figure 1.26, Figure 1.27, Figure 1.28)

• Reasonable water solubility of their succinimidyl esters, permitting conjugations to bedone without addition of organic solvents, if desired

• Chemistry that permits synthesis of singly reactive dyes, thus avoiding crosslinkingreactions

Fluorescence of these long-wavelength Alexa Fluor dyes is not visible to the humaneye but is readily detected by most imaging systems. Pictures of these dyes throughoutthis Handbook have been pseudocolored to represent the staining that is observed withsensitive detection equipment.

An isomeric mixture of the amine-reactive succinimidyl ester of the Alexa Fluor 633dye and the isomer-free succinimidyl esters of the Alexa Fluor 647, Alexa Fluor 660,Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes are available as standalonereagents in either a 1 mg or 5 mg unit size (Table 1.1), and in most cases, as componentsof kits that permit facile labeling of proteins, oligonucleotides and nucleic acids (Table1.1). These kits and their contents are described in detail in Section 1.2.

Alexa Fluor 350 DyeThe sulfonated coumarin derivative, Alexa Fluor 350 carboxylic acid succinimidyl

ester (Figure 1.88), is more water soluble than either AMCA succinimidyl ester orAMCA-X succinimidyl ester (A-6118, Section 1.7) and yields protein conjugates that aremore fluorescent than those prepared from its nonsulfonated analog (Figure 7.28). AlexaFluor 350 protein conjugates are optimally excited at 346 nm (Figure 1.30, Figure 1.89)and have bright-blue fluorescence emission at wavelengths slightly shorter than AMCAor AMCA-X conjugates (442 nm versus 448 nm), which reduces the dye’s spectral over-lap with the emission of fluorescein.

Figure 1.15 Comparison of the absorption andfluorescence emission spectra of the Alexa Fluor555 and Cy3 dyes. Spectra have been normalizedto the same intensity for comparison purposes.

Figure 1.14 Absorption spectra of our intermediate-wavelength light–absorbing Alexa Fluor dyes.

In a practical application of FRET, weprepare tandem conjugates of severallong-wavelength Alexa Fluor dyes withR-phycoerythrin and allophycocyanin.These conjugates, which are describedin Section 6.4, permit multicolor mea-surements using a single laser excita-tion source.

25

TECHNICAL NOTE

Fluorescence Resonance Energy Transfer (FRET)Fluorescence resonance energy transfer (FRET) is a distance-

dependent interaction between the electronic excited states of twodye molecules in which excitation is transferred from a donormolecule to an acceptor molecule without emission of a photon.The efficiency of FRET is dependent on the inverse sixth power ofthe intermolecular separation,1 making it useful over distancescomparable with the dimensions of biological macromolecules.Thus, FRET is an important technique for investigating a variety ofbiological phenomena that produce changes in molecular proximi-ty.2–10 When FRET is used as a contrast mechanism, colocalizationof proteins and other molecules can be imaged with spatial resolu-tion beyond the limits of conventional optical microscopy.11,12

Primary Conditions for FRET• Donor and acceptor molecules must be in close proximity

(typically 10–100 Å).• The absorption spectrum of the acceptor must overlap the

fluorescence emission spectrum of the donor (see figure).• Donor and acceptor transition dipole orientations must be

approximately parallel.

Förster RadiusThe distance at which energy transfer is 50% efficient (i.e., 50%

of excited donors are deactivated by FRET) is defined by the Försterradius (Ro). The magnitude of Ro is dependent on the spectralproperties of the donor and acceptor dyes:

Ro = [8.8 × 1023 • κ2 • n4 QYD • J(λ)]1/6 Å

where κ2 = dipole orientation factor (range 0–4; κ2 = 2⁄3for randomly oriented donors and acceptors

QYD = fluorescence quantum yield of the donor in theabsence of the acceptor

n = refractive indexJ(λ) = spectral overlap integral (see figure)

= �εA(λ) • FD(λ) • (λ)4dλ cm3M-1

where εA = extinction coefficient of acceptorFD = fluorescence emission intensity of donor as a

fraction of the total integrated intensity

Donor/Acceptor PairsIn most applications, the donor and acceptor dyes are different,

in which case FRET can be detected by the appearance of sensitizedfluorescence of the acceptor or by quenching of donor fluores-cence. When the donor and acceptor are the same, FRET can bedetected by the resulting fluorescence depolarization.13 Sometypical values of Ro are listed in the table above and a more exten-sive compilation is in Table 1.3 and Table 1.8. Note that because thecomponent factors of Ro (see above) are dependent on the environ-ment, the actual value observed in a specific experimental situationis somewhat variable. Extensive compilations of Ro values can befound in the literature.4,5,7,10 Nonfluorescent acceptors such asdabcyl and our QSY dyes (Table 1.7) have the particular advantageof eliminating the potential problem of background fluorescenceresulting from direct (i.e., nonsensitized) acceptor excitation.Probes incorporating fluorescent donor–nonfluorescent acceptorcombinations have been developed primarily for detecting proteoly-sis 14 (Figure 10.9) and nucleic acid hybridization 15,16 (Figure 8.101,Figure 8.102).

Selected Applications of FRET• Structure and conformation of proteins 17–22

• Spatial distribution and assembly of protein complexes 23–27

• Receptor/ligand interactions 28–31

• Immunoassays 32,33

• Probing interactions of single molecules 34

• Structure and conformation of nucleic acids 35–40

• Real-time PCR assays and SNP detection 41–46 (Figure 8.103,Figure 8.104)

• Detection of nucleic acid hybridization 15,16,47–50 (Figure 8.101)• Primer-extension assays for detecting mutations 51 (Figure

8.104)• Automated DNA sequencing 52–54

• Distribution and transport of lipids 55–57

• Membrane fusion assays 58–61 (see Lipid-Mixing Assays ofMembrane Fusion in Section 13.2)

• Membrane potential sensing 62

• Fluorogenic protease substrates 14,63–66

• Indicators for cyclic AMP 67,68 (Figure 18.14, Figure 18.15) andzinc 69

Typical Values of R0.

Donor Acceptor Ro (Å)

Fluorescein Tetramethylrhodamine 55

IAEDANS Fluorescein 46

EDANS Dabcyl 33

Fluorescein Fluorescein 44

BODIPY FL BODIPY FL 57

Fluorescein QSY 7 and QSY 9 dyes 61

Schematic representation of the FRET spectral overlap integral.


Section 1.3


References


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Customer Testimonials for the Alexa Fluor Dyes“I have been using Alexa Fluor 594 in place of Texas Red and

have found it to be perhaps five times as sensitive with less back-ground… another investigator in the lab had no results at all untilhe used Alexa Fluor 594 and is now getting excellent results.”

— Warren R. ClarkSenior Biological Scientist

University of FloridaGainesville, Florida USA

“I have been extremely impressed with the quality of the AlexaFluor dyes. They have made it possible to do fluorescent (confocal)analysis on certain antigens that had not previously been possiblewith the standard fluorophores (FITC, rhodamine, Texas Red).”

— Ray GrillAssistant Project Neuroscientist

University of California, San DiegoSan Diego, California USA

“I just tried the Alexa Fluor 488 actin in live tissue culture cells.I was very pleased with the results. On the whole, the labeled actinincorporated into actin stress fibers very well. If anyone asks forfeedback on Alexa Fluor 488 actin, I would be happy to recommendit.”

— Louise CramerGroup Leader, MRC-Laboratory for Molecular Cell Biology

University College LondonLondon, England

“I am using the Alexa Fluor 488 hydrazide salt for intracellularinjections and it is truly great… better than lucifer yellow in bright-ness, photostability and [it exhibits] less bleedthrough to otherchannels… it is by far my first choice.”

— Johan WasséliusDepartment of Ophthalmology

University HospitalLund, Sweden

“I use Alexa Fluor 488, 568 and 594 secondary antibody conju-gates instead of FITC or TRITC because of their great photostabili-ty… staining with Alexa Fluor 488 [provides] the possibility of longtime exposure for serial laser scanning or photomicrography.”

— Olaf AnhennDepartment of Pathology

Ruhr-UniversityBochum, Germany

“All of the Alexa Fluor dyes are superior to anything out there.[They are the] best reagents since sliced bread. FITC has beenbanned from this lab.”

— Joe GoodhouseDepartment of Molecular Biology

Princeton UniversityPrinceton, New Jersey USA

27

Alexa Fluor 430 DyeFew reactive dyes that absorb between 400 nm and 450 nm have appreciable fluores-

cence beyond 500 nm in aqueous solution. Our Alexa Fluor 430 dye fills this spectral gap(Figure 1.30, Figure 1.31). Excitation near its absorption maximum at 431 nm is accom-panied by strong green fluorescence, with an emission maximum at 541 nm. The amine-reactive succinimidyl ester of Alexa Fluor 430 carboxylic acid (A-10169) is available, aswell as Alexa Fluor 430 conjugates of secondary antibodies (A-11063, A-11064; Section7.3) and streptavidin (S-11237, Section 7.6).

Alexa Fluor Labeling Reagents and Kits

All of our Alexa Fluor dyes are available as amine-reactive succinimidyl esters (Table1.1) and most of the Alexa Fluor dyes are also offered as components of several protein andnucleic acid labeling kits (Table 1.1) that are principally discussed in Section 1.2, including:

• Easy-to-Use Protein Labeling Kits (Section 1.2)• Monoclonal Antibody Labeling Kits (Section 1.2)• Zenon One Mouse IgG1 Labeling Kits (Section 7.2; Table 1.1, Table 7.1) for the

easiest and fastest method of making labeled mouse IgG1 monoclonal antibody conju-gates, particularly on a microgram or submicrogram scale

• ARES DNA Labeling Kits (Section 1.2, Section 8.2)• Alexa Fluor Oligonucleotide Amine Labeling Kits (Section 1.2, Section 8.2; Table 8.9)• ULYSIS Nucleic Acid Labeling Kits (Section 8.2, Table 8.7), which utilize Alexa

Fluor conjugates of a guanosine-reactive platinum compound for labeling of intactnucleic acids

These kits and their components are described in detail in the sections and tablesindicated above. In addition, we offer several ChromaTide UTP, ChromaTide dUTP andChromaTide OBEA-dCTP nucleotides (Table 8.5, Table 8.6) that include our Alexa Fluordyes for enzyme-catalyzed incorporation into nucleic acids. The ChromaTide nucleotidesare described in Section 8.2.

Purity of these the Alexa Fluor carboxylic acid succinimidyl esters dyes when preparedand when packaged in a 5 mg unit size (Table 1.1) is usually >80–95% by HPLC. Howev-er, Alexa Fluor dyes tenaciously bind water and packaging of these products in smallerunit sizes — the 1 mg standalone reagents and the multiple vials used in all kits — mayresult in some loss of reactivity. Our specifications for standalone Alexa Fluor carboxylicacid succinimidyl esters that are sold in a 1 mg size or as a component of a labeling kitrequire the product to have reactivity ≥50% after packaging. As part of our quality controlprotocol, we test the suitability of the reactive Alexa Fluor reagents in the 1 mg unit sizeand in all of our Alexa Fluor protein and nucleic acid labeling kits after packaging; how-ever, we recommend that all of the Alexa Fluor carboxylic acid succinimidyl esters andAlexa Fluor protein and nucleic acid labeling kits be used soon after receipt.

Several Alexa Fluor dyes are also available as thiol-reactive maleimides (Section 2.2,Table 2.1) and as aldehyde- and ketone-reactive hydrazides (Section 3.2, Table 3.1). TheAlexa Fluor hydrazides are also important probes for intracellular tracing (Section 14.3;Figure 3.16, Figure 14.22). Some of the Alexa Fluor dyes are mixtures of two isomers;however, all of the Alexa Fluor dyes contain only a single reactive moiety.

The Alexa Fluor fluorophores, reactive dyes, conjugates and their applications are thesubject of several patents and patent applications filed by Molecular Probes, Inc., and areoffered for research purposes only. Molecular Probes welcomes inquiries about licensingthese products for resale or other commercial uses. Custom conjugations of the Alexa Fluorfluorophores are also available. Please contact our Custom and Bulk Sales Department.

Alexa Fluor Bioconjugates and Tandem Conjugates

Alexa Fluor BioconjugatesFor immunofluorescence, receptor labeling, nucleic acid synthesis, cell tracing and

many other applications, we offer Alexa Fluor dyes in a wide variety of bioconjugates,including those of:

Figure 1.16 Neuronal cells in a 22-hour zebrafishembryo were identified with anti–HuC/HuD mousemonoclonal antibody (A-21271) and visualizedwith red-fluorescent Alexa Fluor 568 goat anti–mouse IgG antibody (A-11004). Nuclei werestained with blue-fluorescent DAPI (D-1306,D-3571, D-21490).

Figure 1.17 Photobleaching profiles of the AlexaFluor 555 and Cy3 dyes were obtained by placingequal molar concentrations of the free dyes intocapillary tubes; the samples were continuously illu-minated and data points were collected every fiveseconds. Fluorescence has been normalized to thesame initial intensity.

Section 1.3

As an alternative to directly conjugat-ing primary antibodies with reactivedyes, we strongly recommend use ofthe reagents in our Zenon AntibodyLabeling Kits described in Section 7.2.


Figure 1.19 Brightness comparison of MolecularProbes’ Alexa Fluor 555 goat anti–mouse IgG anti-body with Cy3 goat anti–mouse IgG antibody con-jugates commercially available from several othercompanies. Human blood was blocked with normalgoat serum and incubated with an anti-CD3 mousemonoclonal antibody; cells were washed, resus-pended and incubated with either the Alexa Fluor555 or Cy3 goat anti–mouse IgG antibody at equalconcentrations. Red blood cells were lysed and thesamples were analyzed with a flow cytometerequipped with a 488 nm argon-ion laser and a 585± 21 nm bandpass emission filter.

Figure 1.18 Comparison of the relative fluores-cence of Alexa Fluor 594 and Texas Red-X goatanti–mouse IgG antibody F(ab′)2 fragment conju-gates at different dye:protein ratios.

• Tandem conjugates of R-phycoerythrin (R-PE) and allophycocyanin (APC) for multi-color applications that employ a single laser as an excitation source (Section 6.4;Figure 6.31, Figure 6.34)

• Zenon One labeling reagents (Section 7.2; Table 1.1, Table 7.1), for quick and conve-nient labeling of mouse IgG1 antibodies for multicolor applications

• Secondary antibodies (Section 7.3, Table 7.3)• Protein A and protein G (Section 7.3, Table 7.12)• FluoroNanogold antibody and streptavidin conjugates (Section 7.3, Section 7.6)• Anti-fluorescein/Oregon Green antibody for simultaneously amplifying and photosta-

bilizing the signal of fluorescein- or Oregon Green dye–conjugated probes (A-11090,Section 7.4) and for changing the green fluorescence of these probes to red fluores-cence (A-11091, A-21250; Section 7.4; Figure 7.63, Figure 7.64)

• Anti-biotin and anti–dinitrophenyl-KLH antibodies (Section 7.4)• Primary antibodies, including the CD3, CD4 and CD8 antibodies (Section 7.5)• Anti–green fluorescent protein antibody (anti-GFP; A-21311, A-21312; Section 7.5)• An antibody to the epitope tag hemagglutinin (anti-HA; A-21287, A-21288; Section

7.5)• An antibody to the human HuC/HuD neuronal protein (A-21275, A-21276; Section 7.5)• Anti–glutathione S-transferase antibody (A-11131, Section 7.5)• Avidin and streptavidin (Section 7.6, Table 7.17)• Lectins (Section 7.7, Table 7.18)• UTP, dUTP and OBEA-dCTP nucleotides (Section 8.2) for enzyme-mediated incor-

poration into nucleic acids• Panomer 9 random oligodeoxynucleotide and oligodeoxythymidine-18 (dT18) conju-

gates (Section 8.5, Table 8.13)• Phalloidin, F-actin and DNase I (Section 11.1, Table 11.1)• Endostatin protein and Angiostatin protein (E-23378, A-23376; Section 15.4)• An antibody to glial fibrillary acidic protein (anti-GFAP, Section 11.2)• Anti–OxPhos complex IV antibody (anti–complex I of cytochrome oxidase, Section

12.2)• Dextrans (Section 14.5)• Bovine serum albumin, parvalbumin, soybean trypsin inhibitor, α-crystallin and chol-

era toxin subunit B (Section 14.7)• Anti-bromodeoxyuridine antibody and kits for following cell proliferation and apopto-

sis (Section 15.4, Section 15.5)• Annexin V (Section 15.5)• Fibrinogen and methotrexate (Section 15.6)• Lipopolysaccharides (Section 16.1, Table 16.1)• Acetylated low-density lipoprotein (Section 16.1)• Escherichia coli, Staphylococcus aureus and zymosan A BioParticles, epidermal

growth factor, histones and transferrin (Section 16.1; Table 16.3, Table 16.2)• Angiotensin II, neuromedin C and substance P (Section 16.2)• α-Bungarotoxin (Section 16.2, Table 16.4)• Apamin (Section 16.3)• Calmodulin (Section 18.3)

Alexa Fluor Tandem Conjugates of PhycobiliproteinsWe have conjugated R-phycoerythrin with an Alexa Fluor 610 dye and with our Alexa

Fluor 647 and Alexa Fluor 680 dyes — and in turn conjugated these fluorescent proteinsto antibodies or streptavidin, yielding tandem conjugates that permit simultaneous multi-color labeling and detection of multiple targets with excitation by a single excitationsource — the 488 nm spectral line of the argon-ion laser (Section 6.4, Figure 6.31).Additionally our Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 tandem conju-gates of allophycocyanin can be combined with allophycocyanin or Alexa Fluor 647bioconjugates for multicolor measurements using excitation by the lasers that emit at 633to 650 nm (Figure 6.34). Zenon One labeling reagents for the facile labeling of mouseand rat IgG1 antibodies with the tandem phycobiliproteins dyes are under development(Section 7.2).

The Alexa Fluor dyes were named afterAlexander Haugland and the MarinaBlue dyes after Marina Haugland.

29Section 1.3

Figure 1.20 Fluorescence output from an AlexaFluor 546 goat anti–mouse IgG antibody(dye:protein ratio = 5.7) and a commercially avail-able Cy3 goat anti–mouse IgG antibody(dye:protein ratio = 3.8). Antibody concentrationswere adjusted to give equal absorbance at theexcitation wavelength (535 nm). The relative fluo-rescence quantum yield of Alexa Fluor 546 conju-gates is higher than that of Cy3 conjugates, evenat high dye:protein ratios that would typicallyresult in self-quenching effects with most otherprotein-labeling dyes.

Figure 1.21 Absorption spectra of our long-wave-length light–absorbing Alexa Fluor dyes.

Figure 1.22 Comparison of the fluorescence spec-tra of the Alexa Fluor 647 and Cy5 dyes. Spectrahave been normalized to the same intensity forcomparison purposes.

Figure 1.23 Comparison of the fluorescence spec-tra of the Alexa Fluor 680 and Cy5.5 dyes. Spectrahave been normalized to the same intensity forcomparison purposes.

Figure 1.24 Comparison of the fluorescence emis-sion spectra of the Alexa Fluor 750 and Cy7 dyes.Spectra have been normalized to the same intensi-ty for comparison purposes.

Figure 1.25 Comparison of the relative fluores-cence of goat anti–rabbit IgG antibody conjugatesof the Alexa Fluor 555 and Cy3 dyes (prepared byMolecular Probes, Inc.) at different dye:protein ra-tios in the conjugate.

Figure 1.26 Comparison of the brightness of AlexaFluor 647 and Cy5 dye antibody conjugates (pre-pared by Molecular Probes, Inc.). More Alexa Fluor647 dye molecules can be attached to proteins andnucleic acids without significant quenching, allow-ing for conjugates that are much brighter thanthose possible using the Cy5 dye.


Figure 1.27 Flow cytometry was used to comparethe brightness of Molecular Probes’ Alexa Fluor647 goat anti–mouse IgG antibody (red, A-21235)with commercially available Cy5 goat anti–mouseIgG antibody from Jackson ImmunoResearch Lab-oratories (green) and Amersham-Pharmacia Bio-tech (blue). Human blood was blocked with normalgoat serum and incubated with an anti-CD3 mousemonoclonal antibody; cells were washed, resus-pended and incubated with either an Alexa Fluor647 or Cy5 goat anti–mouse IgG secondary anti-body at equal concentration. Red blood cells werelysed and the samples were analyzed on a flow cy-tometer equipped with a 633 nm He–Ne laser anda long-pass emission filter (>650 nm).

Figure 1.28 Brightness comparison of MolecularProbes’ Alexa Fluor 647 goat anti–mouse IgG anti-body with Cy5 goat anti–mouse IgG antibody con-jugates commercially available from other compa-nies. Human blood was blocked with normal goatserum and incubated with an anti-CD3 mousemonoclonal antibody; cells were washed, resus-pended and incubated with either Alexa Fluor 647or Cy5 goat anti–mouse IgG antibody at an equalconcentration. Red blood cells were lysed and thesamples were analyzed with a flow cytometerequipped with a 633 nm He–Ne laser and a long-pass emission filter (>650 nm).

Signal Amplification with Alexa Fluor Dyes

Tyramide Signal AmplificationTyramide signal-amplification (TSA) technology, which was developed by NEN (now

a part of PerkinElmer Corporation) and licensed to Molecular Probes for in-cell and in-tissue applications, permits significant amplification of cellular targets by a horseradishperoxidase (HRP)–mediated scheme (Figure 6.6). Molecular Probes has introducedseveral TSA Kits (Section 6.2, Table 6.1), including kits that utilize Alexa Fluor 350tyramide (Figure 1.32), Alexa Fluor 488 tyramide (Figure 1.33, Figure 1.34), Alexa Fluor546 tyramide, Alexa Fluor 568 tyramide (Figure 1.35), Alexa Fluor 594 tyramide andAlexa Fluor 647 tyramide (Figure 1.36) as the amplification reagent. The HRP-catalyzedimmobilization of a fluorescent tyramide can yield far greater total fluorescence thanwould ever be possible with direct labeling of the target — enabling detection of verylow-abundance receptors (Figure 6.11) — and can be used in either live- or fixed-cellpreparations. TSA also permits use of greatly decreased quantities of precious antibodiesor nucleic acid probes. Our TSA Kits are listed in Table 6.1 and are extensively discussedin Section 6.2.

Antibody-Based Signal-Amplification KitsAlthough the direct fluorescence signal of Alexa Fluor conjugates tends to be signifi-

cantly greater than that of other dyes with comparable spectra, we have also developedtwo kits that take further advantage of the superior brightness and photostability of AlexaFluor 488 dye– and Alexa Fluor 594 dye–labeled reagents. These Alexa Fluor Signal-Amplification Kits are designed to substantially increase the signals obtained by immu-nofluorescence techniques (Figure 7.53), thus permitting detection of low-abundancetargets. The Alexa Fluor 488 Signal-Amplification Kit for Fluorescein-ConjugatedProbes (A-11053) dramatically enhances the fluorescence and photostability of virtuallyany fluoresceinated probe (Figure 7.52). The Alexa Fluor 488 Signal-Amplification Kitfor Mouse Antibodies (A-11054) can be used to sensitively detect mouse primary anti-bodies. The similar Alexa Fluor 568 and Alexa Fluor 594 Signal-Amplification Kits forMouse Antibodies (A-11066, A-11067) provide ultrasensitive immunofluorescent detec-tion at longer wavelengths. For additional details about these kits, see Section 7.3 and ourproduct literature.

Alexa Fluor Conjugates of Anti-Fluorescein/Oregon Green AntibodyOur Alexa Fluor 488 dye–labeled rabbit anti-fluorescein/Oregon Green antibody

(A-11090, Section 7.4) can be used to enhance the green-fluorescent signal of the fluo-rescein (or Oregon Green) hapten without changing its fluorescence color. Thus, thisconjugate allows researchers to take advantage of the superior photostability of the AlexaFluor 488 dye, while utilizing existing fluorescein- or Oregon Green dye–labeled probesand fluorescein-compatible optics (Table 24.8). The Alexa Fluor 594 dye–labeled rabbitanti-fluorescein/Oregon Green antibody (A-11091) can be used to convert the greenfluorescence of fluorescein or Oregon Green conjugates into exceptionally photostablered fluorescence (Figure 7.63), and to amplify the signal from fluorescein and OregonGreen conjugates by as much as 100-fold (Figure 7.64).

Antibody to the Alexa Fluor 488 DyeWe offer a polyclonal antibody to the Alexa Fluor 488 dye (A-11094, Section 7.4) that

quenches the dye’s fluorescence and can be used in various signal-amplification schemes,potentially including further amplification of the signal from the TSA Kits that containAlexa Fluor 488 tyramide (T-20912, T-20922, T-20932; Section 6.2) or from Alexa Fluorconjugates of proteins or nucleic acids.

References

1. J Histochem Cytochem 47, 1179 (1999); 2. Cytometry 41, 316 (2000); 3. J Bacteriol 182, 2793 (2000);4. J Histochem Cytochem 47, 1213 (1999); 5. J Histochem Cytochem 48, 653 (2000); 6. Bioconjug Chem11, 696 (2000).

31

Figure 1.29 The absorption spectra of the Cy5 dyeconjugates of both proteins and nucleic acidsshow an additional peak at about 600 nm whencompared to the spectrum of the free dye. Howev-er, light absorbed by the Cy5 dye conjugates at thiswavelength does not result in fluorescence. AlexaFluor 647 dye conjugates of proteins do not exhibitthis spectral anomaly. Spectra have been normal-ized to the same peak intensity for comparisonpurposes.

Section 1.3

Figure 1.31 A bovine pulmonary artery endothelial(BPAE) cell labeled with mouse monoclonal anti–α-tubulin antibody (A-11126) in combination withAlexa Fluor 430 goat anti–mouse IgG antibody(A-11063) to stain microtubules. The image wasacquired using a longpass filter set allowing excita-tion at 455 ± 35 nm and emission at wavelengthsgreater than 515 nm.

Figure 1.32 Fixed and permeabilized bovine pul-monary artery endothelial (BPAE) cells labeledwith mouse monoclonal anti–α-tubulin antibody(A-11126) and detected using TSA Kit #7 with theHRP conjugate of goat anti–mouse IgG antibodyand Alexa Fluor 350 tyramide (T-20912).

Figure 1.33 A zebrafish retina cryosection labeledwith the mouse monoclonal antibody FRet 6 anddetected using TSA Kit #9 with the HRP conjugateof goat anti–mouse IgG antibody and Alexa Fluor488 tyramide (T-20912). Figure 1.34 A zebrafish retina cryosection labeled

with the mouse monoclonal antibody FRet 43 anddetected using TSA Kit #9 with the HRP conjugateof goat anti–mouse IgG antibody and green-fluo-rescent Alexa Fluor 488 tyramide (T-20912). Thenuclei were counterstained with blue-fluorescentHoechst 33258 (H-1398, H-3569, H-21491).

Figure 1.30 Absorption spectra of our short-wave-length light–absorbing Alexa Fluor dyes.


TECHNICAL NOTE

Fluorescence Correlation Spectroscopy (FCS)intensity is recorded for a small number of molecules in the detec-tion volume (e.g., 3 molecules/femtoliter, equivalent to ~5 nMmacroscopic concentration) over a time range from about 1 micro-second to 1 second. The time-dependent fluorescence intensity(F(t)) is then analyzed in terms of its temporal autocorrelationfunction (G (τ)), which compares the fluorescence intensity at timet with the intensity at (t + τ), where τ is a variable interval, averagedover all data points in the time series (denoted by < >):

The autocorrelation function contains information about equilib-rium concentrations, reaction kinetics and diffusion rates of mole-cules in the sample. The initial amplitude of the autocorrelationfunction is inversely proportional to number of molecules in thedetection volume. The autocorrelation function decays from itsinitial value with a time-dependence that is determined by molecu-lar diffusion rates. For example, free fluorescent ligands exhibitfaster autocorrelation decay than slower-moving complexed ligands(Figure 2).


Figure 1 Physical origins of fluorescence correlation spectroscopy data.Free fluorescent ligands move in and out of the detection volume (open cir-cle) and are detected as a series of short, randomized fluorescence bursts(top panel). Macromolecule-bound ligands are less mobile, producing amore slowly fluctuating (i.e., more highly autocorrelated) time-dependentfluorescence pattern (bottom panel).

Figure 2 Simulated FCS autocorrelation functions representing a low molec-ular weight ligand (left curve, blue), macromolecule-bound ligand (rightcurve, red) and a 1:1 mixture of free and bound ligand (middle curve, green).

Fluorescence correlation spectroscopy (FCS) is a technique inwhich spontaneous fluorescence intensity fluctuations are mea-sured in a microscopic detection volume of about 10–15 L (1 femto-liter) defined by a tightly focused laser beam.1,2 Renewed interest inFCS in recent years has been stimulated by the fact that it is inher-ently miniaturized and therefore applicable for high-throughputscreening applications.3 Fluorescence intensity fluctuations mea-sured by FCS represent changes in either the number or the fluo-rescence quantum yield of molecules resident in the detectionvolume (Figure 1). Small, rapidly diffusing molecules producerapidly fluctuating intensity patterns, whereas larger moleculesproduce more sustained bursts of fluorescence.

This situation is in marked contrast to conventional fluores-cence photometry carried out in sample volumes of around0.1–1.0 mL (~108 times larger than FCS measurement volumes)that report only the macroscopic average of diffusion-dependentintensity fluctuations. In a typical FCS measurement, fluorescence

Probes and Applications for FCSFCS is applicable for monitoring a multitude of biomolecular

association and dissociation processes (see Table below). BecauseFCS is intrinsically sensitive to the mass changes occurring in theseprocesses, probe design and selection is generally less critical thanit is in assays based on macroscopic fluorescence intensity chang-es generated by dye–dye interactions (FRET, self-quenching, etc.)or environment-dependent fluorescence enhancement. Dyes thatperform well in confocal laser-scanning microscopy are usuallyamong the best choices for FCS applications. Laser sources usedfor excitation in FCS include the argon-ion 488 nm spectral line, theHe-Ne 543 nm and 633 nm spectral lines and the argon/krypton-ion568 nm and 647 nm spectral lines. Dyes with appreciable rates of

33


Detected Process References

Nucleic acid fragmentation Anal Biochem 260, 166 (1998); Proc Natl Acad Sci U S A 95, 1421 (1998); Proc Natl Acad Sci U S A 95,1416 (1998)

Nucleic acid hybridization Biochemistry 35, 10182 (1996); Nucleic Acids Res 23, 1795 (1995)

PCR product formation Biochemistry 37, 12971 (1998); Biotechniques 25, 706 (1998); Proc Natl Acad Sci U S A 93, 12805(1996)

Lateral segregation of lipids in bilayermembranes

Cytometry 36, 176 (1999); Proc Natl Acad Sci U S A 96, 8461 (1999)

Molecular diffusion in the nucleus andcytoplasm

Biophys J 75, 2547 (1998); Proc Natl Acad Sci U S A 95, 6043 (1998)

Protein–protein interactions Biochem Biophys Res Commun 267, 300 (2000); Biochemistry 38, 13759 (1999); Biochemistry 38,8402 (1999); Chem Biol 6, 53 (1999); Cytometry 36, 247 (1999); Biophys Chem 75, 151 (1998)

Binding equilibria for drugs and other lowmolecular weight ligands

Biochemistry 38, 5082 (1999); Biochemistry 38, 8671 (1999); J Biomol Screen 4, 355 (1999);Biophys J 73, 2195 (1997); Biophys Chem 58, 3 (1996)

Clustering of membrane-bound receptors Biophys J 70, 2001 (1996); Biophys J 65, 1135 (1993); Chem Phys Lipids 50, 253 (1989)

Applications of fluorescence correlation spectroscopy.

triplet state population via intersystem crossing are generally notwell suited for FCS measurement because this process results in anadditional sub-millisecond autocorrelation decay component.4

Recent Technical Developments in FCSTwo-photon excitation (TPE) has been applied to FCS for rea-

sons similar to those that have motivated its use in fluorescencemicroscopy — inherent spatial confinement of excitation, dimin-ished photobleaching and photoxicity, less scattering and betteroptical penetration in turbid media.5,6 Dual-color cross-correlationFCS 7 measures the cross-correlation of the time-dependent fluo-rescence intensities of two spectrally distinct dyes, instead of theconventional autocorrelation for a single dye. This approach has theadvantage that cross-correlated fluorescence is only generated bymolecules or complexes labeled with both dyes, allowing quantita-

tion of interacting molecules without reference to their diffusioncharacteristics. In practice, discrimination on the basis of mass inconventional FCS requires that the interacting components shouldhave a molecular weight ratio of at least 1:7. FCS measurementsusing TPE in combination with dual-color cross-correlation haverecently been reported for the first time.8

References

1. Proc Natl Acad Sci U S A 94, 11753 (1997); 2. Topics in FluorescenceSpectroscopy, Lakowicz JR, Ed. 1: Techniques, 337 (1991); 3. J BiomolScreen 4, 335 (1999); 4. J Phys Chem 99, 13368 (1995); 5. Biophys J 77,2251 (1999); 6. Biophys J 71, 410 (1996); 7. Biophys J 72, 1878 (1997);8. Proc Natl Acad Sci U S A 97, 10377 (2000).

Section 1.3

Figure 1.35 Fixed and permeabilized bovine pul-monary artery endothelial cells (BPAEC) labeledwith anti–OxPhos Complex IV subunit I (human)antibody (anti–cytochrome oxidase subunit I)(A-6403) and detected using TSA Kit #4 with theHRP conjugate of goat anti–mouse IgG antibodyand Alexa Fluor 568 tyramide (T-20914).

Figure 1.36 Fixed and permeabilized bovine pulmonary artery endothelial cells (BPAEC) labeled with anti–OxPhos Complex IV subunit I (human) antibody (anti–cytochrome oxidase subunit I) (A-6403) and detectedusing TSA Kit #6 with the HRP conjugate of goat anti–mouse IgG antibody and Alexa Fluor 647 tyramide(T-20916). The image was deconvolved using Huygens software (Scientific Volume Imaging, www.svi.nl).


Data Table — 1.3 Alexa Fluor Dyes: Simply the Best

Cat # MW Storage Soluble Abs EC Em Solvent NotesA-10168 410.35 F,D,L H2O, DMSO 346 19,000 445 pH 7 1A-10169 701.75 F,D,L H2O, DMSO 430 15,000 545 pH 7 1A-20000, A-20100 643.41 F,DD,L H2O, DMSO 494 78,000 517 pH 7 1, 2A-20001, A-20101 723.77 F,DD,L H2O, DMSO 530 81,000 555 pH 7 1A-20002, A-20102 1079.39 F,DD,L H2O, DMSO 554 112,000 570 pH 7 1A-20003, A-20103 791.80 F,DD,L H2O, DMSO 578 88,000 602 pH 7 1, 3A-20004, A-20104 819.85 F,DD,L H2O, DMSO 590 92,000 617 pH 7 1, 4A-20005, A-20105 ~1200 F,DD,L H2O, DMSO 621 159,000 639 MeOH 1, 5, 6A-20006, A-20106 ~1250 F,DD,L H2O, DMSO 649 250,000 666 pH 7 1A-20007, A-20107 ~1100 F,DD,L H2O, DMSO 668 132,000 698 MeOH 1, 7A-20008, A-20108 ~1150 F,DD,L H2O, DMSO 684 183,000 707 MeOH 1, 8A-20009, A-20109 ~1250 F,DD,L H2O, DMSO 555 158,000 571 MeOH 1A-20010, A-20110 ~1400 F,DD,L H2O, DMSO 702 196,000 723 MeOH 1A-20011, A-20110 ~1300 F,DD,L H2O, DMSO 749 240,000 782 MeOH 1

For definitions of the contents of this data table, see “How to Use This Book” on page viii.

Notes1. This sulfonated succinimidyl ester derivative is water soluble and may be dissolved in buffer at ~pH 8 for reaction with amines. Long-term storage in water is NOT recommended due

to hydrolysis.2. Fluorescence lifetime (τ) of Alexa Fluor 488 dye in pH 7.4 buffer at 20°C is 4.1 nanoseconds. Data provided by the SPEX Fluorescence Group, Jobin Yvon, Inc.3. Fluorescence lifetime (τ) of Alexa Fluor 568 dye in pH 7.4 buffer at 20°C is 3.6 nanoseconds. Data provided by the SPEX Fluorescence Group, Jobin Yvon, Inc.4. Fluorescence lifetime (τ) of Alexa Fluor 594 dye in pH 7.4 buffer at 20°C is 3.9 nanoseconds. Data provided by the SPEX Fluorescence Group, Jobin Yvon, Inc.5. Alexa Fluor 633 dye–labeled proteins typically exhibit two absorption peaks at about ~580 and ~630 nm. Fluorescence excitation is more efficient at the 630 nm absorption peak.6. Fluorescence lifetime (τ) of Alexa Fluor 633 dye in H2O at 20°C is 3.2 nanoseconds. Data provided by LJL BioSystems/Molecular Devices Corporation.7. Fluorescence lifetime (τ) of Alexa Fluor 660 dye in aqueous buffer (pH 7.5) at 20°C is 1.2 nanoseconds. Data provided by Pierre-Alain Muller, Max Planck Institute for Biophysical

Chemistry, Göttingen.8. Fluorescence lifetime (τ) of Alexa Fluor 680 dye in aqueous buffer (pH 7.5) at 20°C is 1.2 nanoseconds. Data provided by Pierre-Alain Muller, Max Planck Institute for Biophysical

Chemistry, Göttingen.

Product List — 1.3 Alexa Fluor Dyes: Simply the Best

Cat # Product Name Unit SizeA-10168 Alexa Fluor® 350 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-10169 Alexa Fluor® 430 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20000 Alexa Fluor® 488 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20100 Alexa Fluor® 488 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20001 Alexa Fluor® 532 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20101 Alexa Fluor® 532 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20002 Alexa Fluor® 546 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20102 Alexa Fluor® 546 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20009 Alexa Fluor® 555 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20109 Alexa Fluor® 555 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20003 Alexa Fluor® 568 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20103 Alexa Fluor® 568 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20004 Alexa Fluor® 594 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20104 Alexa Fluor® 594 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20005 Alexa Fluor® 633 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20105 Alexa Fluor® 633 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20006 Alexa Fluor® 647 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20106 Alexa Fluor® 647 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20007 Alexa Fluor® 660 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20107 Alexa Fluor® 660 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20008 Alexa Fluor® 680 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20108 Alexa Fluor® 680 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20010 Alexa Fluor® 700 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20110 Alexa Fluor® 700 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20011 Alexa Fluor® 750 carboxylic acid, succinimidyl ester ............................................................................................................................................ 1 mgA-20111 Alexa Fluor® 750 carboxylic acid, succinimidyl ester ............................................................................................................................................ 5 mgA-20181 Alexa Fluor® 488 Monoclonal Antibody Labeling Kit *5 labelings* ....................................................................................................................... 1 kitA-20182 Alexa Fluor® 532 Monoclonal Antibody Labeling Kit *5 labelings* ....................................................................................................................... 1 kitA-20183 Alexa Fluor® 546 Monoclonal Antibody Labeling Kit *5 labelings* ....................................................................................................................... 1 kitA-20187 Alexa Fluor® 555 Monoclonal Antibody Labeling Kit *5 labelings* ....................................................................................................................... 1 kitA-20184 Alexa Fluor® 568 Monoclonal Antibody Labeling Kit *5 labelings* ....................................................................................................................... 1 kitA-20185 Alexa Fluor® 594 Monoclonal Antibody Labeling Kit *5 labelings* ....................................................................................................................... 1 kitA-20186 Alexa Fluor® 647 Monoclonal Antibody Labeling Kit *5 labelings* ....................................................................................................................... 1 kit

35Section 1.3

Custom ConjugationsMolecular Probes routinely prepares quality bioconjugates with

antibodies and many other biomolecules, using our proprietaryand standard dyes. We can also custom conjugate your antibodyor other protein to biotin, enzymes or other molecules, and we canprepare custom conjugates of our proprietary Alexa Fluor, OregonGreen, BODIPY, Texas Red-X, Rhodamine Red-X, Marina Blue,Pacific Blue, Cascade Blue and QSY dyes.* Our service is efficientand confidential, and we guarantee our work. Molecular Probes isISO certified and is experienced at performing both small- and

large-scale conjugations and purifications. Molecular Probes isalso an O.E.M. supplier of a variety of protein conjugates andfluorescent reagents.

For more information about the availability and cost of customconjugations, visit our Web site (www.probes.com/probes/custom/conjugations.html).

* Use of custom conjugates that include these dyes is restrictedto internal research and development — resale of the conjugates oruse for fee-for-service activities is prohibited; however, licensesmay be available. For licensing information, [email protected].

A-20191 Alexa Fluor® 488 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20192 Alexa Fluor® 532 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20193 Alexa Fluor® 546 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20197 Alexa Fluor® 555 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20194 Alexa Fluor® 568 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20195 Alexa Fluor® 594 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20196 Alexa Fluor® 647 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-10170 Alexa Fluor® 350 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-10171 Alexa Fluor® 430 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-10235 Alexa Fluor® 488 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-10236 Alexa Fluor® 532 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-10237 Alexa Fluor® 546 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-20174 Alexa Fluor® 555 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-10238 Alexa Fluor® 568 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-10239 Alexa Fluor® 594 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-20170 Alexa Fluor® 633 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-20173 Alexa Fluor® 647 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-20171 Alexa Fluor® 660 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-20172 Alexa Fluor® 680 Protein Labeling Kit *3 labelings* ............................................................................................................................................. 1 kitA-21665 ARES™ Alexa Fluor® 488 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21666 ARES™ Alexa Fluor® 532 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21667 ARES™ Alexa Fluor® 546 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21677 ARES™ Alexa Fluor® 555 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21668 ARES™ Alexa Fluor® 568 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21669 ARES™ Alexa Fluor® 594 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21676 ARES™ Alexa Fluor® 647 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21671 ARES™ Alexa Fluor® 660 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21672 ARES™ Alexa Fluor® 680 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitU-21650 ULYSIS® Alexa Fluor® 488 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21651 ULYSIS® Alexa Fluor® 532 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21652 ULYSIS® Alexa Fluor® 546 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21653 ULYSIS® Alexa Fluor® 568 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21654 ULYSIS® Alexa Fluor® 594 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21660 ULYSIS® Alexa Fluor® 647 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21656 ULYSIS® Alexa Fluor® 660 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21657 ULYSIS® Alexa Fluor® 680 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitZ-25000 Zenon™ One Alexa Fluor® 350 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25001 Zenon™ One Alexa Fluor® 430 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25002 Zenon™ One Alexa Fluor® 488 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25003 Zenon™ One Alexa Fluor® 532 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25004 Zenon™ One Alexa Fluor® 546 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25006 Zenon™ One Alexa Fluor® 568 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25007 Zenon™ One Alexa Fluor® 594 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25008 Zenon™ One Alexa Fluor® 647 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25009 Zenon™ One Alexa Fluor® 660 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25010 Zenon™ One Alexa Fluor® 680 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25011 Zenon™ One Alexa Fluor® 700 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kitZ-25012 Zenon™ One Alexa Fluor® 750 Mouse IgG1 Labeling Kit *50 labelings* ............................................................................................................. 1 kit

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