Hermanson G. Bioconjugate Techniques, Second Edition

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450 9. Fluorescent Probes

The spectral characteristics of BODIPY FL IA somewhat mimic the green luminescence of ﬂ u-

orescein, thus the FL designation in its name. The excitation maximum for the probe occurs at

504 nm and its emission at 510 nm. The extremely small Stoke ’s shift makes it difﬁ cult to avoid

problems of excitation-light interference in critical emission measurements unless sub-optimal

excitation wavelengths below its excitation maximum are used. The molecule has an extinction

coefﬁ cient in methanol (containing 1 percent sodium acetate and 1 percent 2-mercaptoethanol)

of about 79,000 M

 1

at 504 nm.

BODIPY FL IA is insoluble in aqueous solution, but may be dissolved in DMF or DMSO

as a concentrated stock solution prior to addition of a small aliquot to a reaction mixture.

Coupling to sulfhydryl-containing molecules occurs rapidly with the formation of a thioether

linkage. The reaction may be done in 50 mM sodium borate, 5 mM EDTA, pH 8.3. The main

consideration is to protect the iodoacetyl derivative from light which may generate iodine and

Figure 9.34 The long side chain of this BODIPY derivative contains a sulfhydryl-reactive iodoacetamide group

that can couple to a thiol group to form a thioether bond.

Figure 9.35 The iodoacetamide group of this BODIPY ﬂ uorophore can react with sulfhydryl-containing mol-

ecules to form thioether linkages.

reduce the reactivity of the probe. In addition, to avoid the ﬂ uorescence quenching effects that

are often a problem with BODIPY probes, react no more than a 2- to 4-fold molar excess of

probe to the amount of sulfhydryl groups present. Oligonucleotides containing a sulfhydryl

modiﬁ cation at their 5  ends (Chapter 27, Section 2.2) may be coupled with BODIPY FL IA,

yielding highly ﬂ uorescent probes.

BODIPY 530/550 IA

BODIPY 530/550 IA is N-(4,4-diﬂ uoro-5,7-diphenyl-4-bora-3a,4a-diaza- s-indacene-3-propionyl)-

N -iodoacetylethylenediamine, a derivative similar to that of BODIPY FL IA, but containing two

phenyl groups rather than two methyl substituents in the No. 5 and 7 positions. This change in

structure results in modulation of the spectral characteristics such that its excitation and emission

wavelengths and its Stoke ’s shift are all increased. The spacer arm off the No. 3 carbon atom of

the basic BODIPY core contains a terminal iodoacetyl group, which reacts with sulfhydryl groups

in proteins and other macromolecules to create stable thioether linkages ( Figure 9.35 ).

The excitation maximum for BODIPY 530/550 IA occurs at 534 nm and its emission at

552 nm. The Stoke ’s shift is greater than the “ FL ” BODIPY derivatives, having an 18 nm differ-

ential between excitation and emission wavelengths. The molecule has an extinction coefﬁ cient

in methanol (containing 1 percent sodium acetate and 1 percent 2-mercaptoethanol) of about

69,000 M

 1

at 534 nm.

BODIPY 530/550 IA is insoluble in aqueous solution, but may be dissolved in DMF or

DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction

mixture. Coupling to sulfhydryl-containing molecules occurs rapidly with the formation of a

thioether linkage. The reaction may be done in 50 mM sodium borate, 5 mM EDTA, pH 8.3.

4. BODIPY Derivatives 451

452 9. Fluorescent Probes

The main consideration is to protect the iodoacetyl derivative from light which may gener-

ate iodine and reduce the reactivity of the probe. To limit the degree of ﬂ uorescent quenching

in the resultant conjugate, the probe should be reacted at no more than a 2- to 4-fold molar

excess over the amount of target molecule present.

Br-BODIPY 493/503

Br-BODIPY 493/503 is 8-bromomethyl-4,4-diﬂ uoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-

3-indacene, a small BODIPY derivative containing a short, sulfhydryl-reactive bromomethyl

group. The modiﬁ cations to the core molecule result in modulation of its spectral characteris-

tics such that, after conjugation, its excitation and emission wavelengths are reduced somewhat

from other BODIPY probes. The reagent can be coupled to  SH containing molecules to pro-

duce a thioether linkage ( Figure 9.36 ).

The excitation maximum for Br-BODIPY 493/503 is 515 nm and its emission occurs at

525 nm when dissolved in methanol. Upon coupling to a sulfhydryl compound, however, the

excitation wavelength of the adduct decreases to 493 nm and its emission drops to 503 nm. The

very small 10 nm Stoke ’s shift may be a problem, particularly in avoiding interference due to of

excitation-light scattering in critical emission measurements. Sub-optimal excitation wavelengths

Figure 9.36 Br-BODIPY can be used to modify sulfhydryl-containing molecules to form thioether linkages.

below the excitation maximum may be used to reduce extraneous light contamination. The mol-

ecule has an extinction coefﬁ cient in methanol of about 55,000 M

 1

at 515 nm.

Br-BODIPY 493/503 is insoluble in aqueous reaction mixtures, but may be dissolved in

DMF or DMSO as a concentrated stock solution prior to addition of a small amount to a buff-

ered solution. Coupling to sulfhydryl-containing molecules is rapid, leading to the formation of

a thioether linkage. The reaction may be done in 50 mM sodium borate, 5 mM EDTA, pH 8.3.

An important consideration is to protect the iodoacetyl derivative from light which may gener-

ate iodine and reduce the reactivity of the probe.

5. Cascade Blue Derivatives

Cascade Blue derivatives are ﬂ uorescent probes having strong luminescence in the blue region

of the spectrum (Invitrogen). The basic Cascade Blue molecule is derived from a trisulfonated

pyrene backbone ( Figure 9.37 ) (Whitaker et al., 1991). It is a ﬁ xable analog of 8-methox-

ypyrene-1,3,6-trisulfonic acid, which is a blue ﬂ uorescent neural tracer. The ﬂ uorophore emits

light in a region removed form the luminescent signal of ﬂ uorescein or Lucifer Yellow, making

it a good choice for multi-labeling applications. The dye can be used along with Lucifer Yellow

CH and sulforhodamine 101 for three-color mapping of neuronal components and processes.

Cascade Blue derivatives have relatively high absorptivity, good QY (typically about 0.54),

excellent water solubility due to the presence of the negatively charged sulfonate groups, and

good photostability. Labeling proteins and other macromolecules with Cascade Blue deriva-

tives can be done with little ﬂ uorescent quenching due to dye–dye interactions.

The following sections discuss the most important Cascade Blue derivatives that are avail-

able for covalent modiﬁ cation purposes.

Amine-Reactive: Cascade Blue Acetyl Azide

One Cascade Blue derivative is available for creating linkages with amine-containing molecules.

The acetyl azide functionality of this reagent reacts with primary amines at ambient tempera-

tures or below to create amide bond derivatives (Lanier and Recktenwald, 1991; Oparka et al.,

Figure 9.37 The basic structure of Cascade Blue ﬂ uorophores.

5. Cascade Blue Derivatives 453

454 9. Fluorescent Probes

1991). At elevated temperatures (80 °C in DMF), the acetyl azide group rearranges to form

an isocyanate that can react with hydroxyl-containing molecules to form a urethane linkage

(Figure 9.38 ). The Cascade Blue urethane derivatives of macromolecules are extremely ﬂ uores-

cent and can be detected down to femtogram quantities (Takadate et al., 1985).

Figure 9.38 The acetyl azide group of this Cascade Blue derivative has dual functions. It can react with amine

groups to form amide bonds, or it can be converted to an isocyanate at high temperatures to couple with

hydroxyl functional groups, creating a carbamate linkage.

This ﬂ uorophore has excitation maxima at 375 and 400 nm and an emission maximum at

410 nm. The small Stoke ’s shift may create some difﬁ culty in discrete excitation without con-

taminating the emission measurement with scattered or overlapping light. The extinction coef-

ﬁ cient of the molecule in water is about 27,000 M

 1

. Cascade Blue and Lucifer Yellow

derivatives can be simultaneously excited by light of less than 400 nm, resulting in two-color

detection at 410 and 530 nm.

Cascade Blue acetyl azide is soluble in aqueous solution, but the reactive azide group will

hydrolyze and should be used immediately in a conjugation reaction. A concentrated stock

solution may be prepared in water, dissolved quickly, and an aliquot quickly added to a buff-

ered reaction medium. For aqueous reactions, a pH range of 7–9 is optimal. Avoid amine-

containing buffers.

Carboxylate-Reactive: Cascade Blue Cadaverine and Cascade Blue Ethylenediamine

Cascade Blue cadaverine and Cascade Blue ethylenediamine both contain a carboxamide-

linked diamine spacer off the 8-methoxy group of the pyrene trisulfonic acid backbone.

The cadaverine version contains a 5-carbon spacer, while the ethylenediamine compound has

only a 2-carbon arm. Both can be coupled to carboxylic acid-containing molecules using a

carbodiimide reaction (Chapter 3, Section 1). Since Cascade Blue derivatives are water-solu-

ble, the carbodiimide EDC can be used to couple these ﬂ uorophores to proteins and other

carboxylate-containing molecules in aqueous solutions at a pH range of 4.5–7.5. The reaction

forms amide bond linkages ( Figure 9.39 ).

These ﬂ uorophores have excitation maxima at 377–378 nm and at 398–399 nm and

emission maxima at 422–423 nm. The extinction coefﬁ cients of the molecules in water are

about 27,000 M

 1

. The Cascade Blue derivatives can be used along with Lucifer Yellow

5. Cascade Blue Derivatives 455

456 9. Fluorescent Probes

derivatives and simultaneously excited by light of less than 400 nm, resulting in two-color

detection at 422 and 530 nm.

Cascade Blue diamine derivatives are soluble in aqueous solution. A concentrated stock

solution may be prepared in water, dissolved quickly, and an aliquot immediately added to a

buffered reaction medium. For aqueous reactions, 0.1 M MES, pH 4.7–6.5, may be used to sta-

bilize the pH during the coupling process. Avoid amine- or carboxylate-containing buffers such

as Tris or glycine, since these can compete with the coupling reaction.

Aldehyde/Ketone-Reactive: Cascade Blue Hydrazide

Cascade Blue hydrazide is a carboxy-hydrazine derivative of the 8-methoxy group on the

pyrene trisulfonic acid ﬂ uorophore. Hydrazide groups react with aldehyde and ketone groups

to form relatively stable hydrazone linkages (Chapter 2, Section 5.1) ( Figure 9.40 ). Although

most biomolecules don ’t contain aldehyde or ketone groups in their native state, carbohydrates,

glycoproteins, RNA, and other molecules that contain sugar residues can be oxidized with

Figure 9.39 The side-chain primary amine group of this Cascade Blue derivative can be coupled to carboxylate-

containing molecules using a carbodiimide reaction.

Figure 9.40 Cascade Blue hydrazide can be used to modify aldehyde-containing molecules to form hydrazone

bonds.

sodium periodate to produce reactive formyl groups. The use of modiﬁ cation reagents which

generate aldehydes upon coupling to a molecule also can be used to produce hydrazide-reactive

sites (Chapter 1, Section 4.4).

In addition, DNA and RNA may be modiﬁ ed with hydrazide-reactive probes by reacting their

cytosine residues with bisulﬁ te to form reactive sulfone intermediates (Chapter 27, Section 2.1).

These derivatives can undergo transamination reactions with hydrazide- or amine-containing

probes to yield covalent bonds (Draper and Gold, 1980).

This ﬂ uorophore has an excitation maximum at 400 nm and an emission maximum

at 420 nm. The extinction coefﬁ cient of the molecule in aqueous solution at pH 7 is about

31,000 M

 1

. Cascade Blue hydrazide and Lucifer Yellow derivatives can be excited simul-

taneously by light of less than 400 nm, resulting in two-color detection at 420 and 530 nm.

Cascade Blue hydrazide is soluble in aqueous solution, and it should be stable for awhile if

protected from light. A concentrated stock solution of the reagent may be prepared in water

and an aliquot added to a buffered reaction medium to facilitate the transfer of small quanti-

ties. For aqueous reactions, a pH range of 5–9 will result in efﬁ cient hydrazone formation.

6. Lucifer Yellow Derivatives

Lucifer Yellow derivatives are used extensively for cytochemical staining applications, especially in

neurophysiology (Stewart, 1981a, b). The ﬂ uorophores are 3,6-disulfonate 4-aminonaphthalimide

derivatives that can be further modiﬁ ed at their imide nitrogen to contain reactive groups suit-

able for conjugation with biomolecules ( Figure 9.41 ). Cell staining with membrane-impermeant

Lucifer Yellow dyes is usually done by osmotic shock, microinjection, or pinocytosis (Swanson

et al., 1987). Derivatives containing amines or hydrazide groups are ﬁ xable with formaldehyde

or glutaraldehyde, coupling to nearby proteins or other amine-containing molecules intracel-

lularly during the reaction. After periodate oxidation, glycoconjugates on cell surfaces or in

solution may be labeled with the hydrazide derivative (Stewart, 1978). Sulfhydryl-containing

molecules may be tagged with the iodoacetamide derivative.

6. Lucifer Yellow Derivatives 457

458 9. Fluorescent Probes

Figure 9.41 The basic structure of Lucifer Yellow ﬂ uorophores.

Lucifer Yellow probes are water-soluble to at least 1.5%. The absorbance maximum of

the derivatives occurs about 426–428 nm with an emission peak at about 530–535 nm, in the

yellow region of the spectrum. The QY of Lucifer dyes is about 0.25. The good intensity of

luminosity from these dyes makes possible detection of small quantities of labeled molecules

intracellularly. The ﬂ uorescent conjugates are readily visible in living cells at concentrations

that are nontoxic to cell viability. The low molecular weight and water solubility of these dyes

allow passage of labeled compounds from one cell to another, potentially revealing molecular

relationships between cells.

Sulfhydryl Reactive: Lucifer Yellow Iodoacetamide

One Lucifer Yellow derivative is available for labeling sulfhydryl-containing molecules. Lucifer

Yellow iodoacetamide is a 4-ethyliodoacetamide derivative of the basic disulfonate aminonaph-

thalimide ﬂ uorophore structure (Invitrogen). The iodoacetyl groups react with  SH groups in

proteins and other molecules to form stable thioether linkages ( Figure 9.42 ).

The spectral characteristics of Lucifer Yellow iodoacetamide produce luminescence at some-

what higher wavelengths than the green luminescence of ﬂ uorescein, thus the yellow designa-

tion in its name. The excitation maximum for the probe occurs at 426 nm and its emission at

530 nm. The rather large Stoke ’s shift makes sensitive measurements of emission intensity pos-

sible without interference by scattered excitation light. The 2-mercaptoethanol derivative of the

ﬂ uorophore has an extinction coefﬁ cient at pH 7 of about 13,000 M

 1

at 426 nm.

Lucifer Yellow iodoacetamide is soluble in aqueous solution due to its negatively charged

sulfonate groups. A concentrated stock solution may be prepared in water prior to addition

of a small aliquot to a reaction mixture. Coupling to sulfhydryl-containing molecules occurs

rapidly with the formation of a thioether linkage. The reaction may be done in 50 mM sodium

borate, 5 mM EDTA, pH 8.3. The main consideration is to protect the iodoacetyl derivative

from light, which will generate iodine and reduce the reactivity of the probe. The reaction may

be limited to sulfhydryls (avoiding any amine derivatization) by reacting a low molar excess of

probe to the amount of sulfhydryl groups present. In addition, oligonucleotides containing a

sulfhydryl modiﬁ cation at their 5  ends (Chapter 27, Section 2.2) may be coupled with Lucifer

Yellow iodoacetamide, yielding highly ﬂ uorescent, yellow probes.

Aldehyde/Ketone Reactive: Lucifer Yellow CH

Lucifer Yellow CH is a carbohydrazide derivative of the basic disulfonate aminonaphthalimide

ﬂ uorophore structure (Invitrogen). Hydrazide groups react with aldehyde and ketone groups to

form relatively stable hydrazone linkages (Chapter 2, Section 5.1) ( Figure 9.43 ). Although most

biomolecules don ’t contain aldehyde or ketone groups in their native state, carbohydrates, glyc-

oproteins, RNA, and other molecules that contain sugar residues can be oxidized with sodium

periodate to produce reactive formyl groups. The use of modiﬁ cation reagents which gener-

ate aldehydes upon coupling to a molecule also can be used to produce hydrazide-reactive sites

(Chapter 1, Section 4.4). In addition, DNA and RNA may be modiﬁ ed with hydrazide-reactive

probes by reacting their cytosine residues with bisulﬁ te to form reactive sulfone intermediates

Figure 9.42 Lucifer Yellow iodoacetamide can be used to label sulfhydryl-containing molecules, forming

thioether bonds.

6. Lucifer Yellow Derivatives 459