Hermanson G. Bioconjugate Techniques, Second Edition

Подождите немного. Документ загружается.

400 9. Fluorescent Probes

these dyes and ﬂ uorophores is the presence of the oxygen bridge between the upper phenyl rings

which constricts the molecule to a planer shape, thus conferring luminescent qualities.

Fluorescent compounds are sensitive to changes in their chemical environment. Alterations

in media pH, buffer components, solvent polarity, or dissolved oxygen can affect and quench

the QY of a ﬂ uorescent probe (Bright, 1988). The presence of absorbing components in solu-

tion which absorb light at or near the excitation wavelength of the ﬂ uorophore will have the

effect of decreasing luminescence. In addition, noncovalent interactions of the probe with

other components in solution can inhibit rotational freedom and quench ﬂ uorescence. In this

regard, the binding of an anti-ﬂ uorescein antibody to the ﬂ uorescent dye completely abolishes

its ﬂ uorescence.

Over-labeling with a ﬂ uorescent probe also causes decreases in QY. Quenching caused by

interactions between ﬂ uorescent molecules often occurs as the level of probe substitution reaches

about 8–10 ﬂ uorophores per protein. Not only can the emission intensity decrease severely at

high substitution levels, but the degree of nonspeciﬁ c binding caused by the number of aro-

matic groups attached to the biomolecule can increase severely. Fluorophore self-quenching

at high substitution or concentration levels can be due to energy transfer from excited-state

molecules to ground-state dimers (Chen and Knutson, 1988).

One method of reducing dye–dye interactions is to add negative charge character to the ﬂ uo-

rescent molecule. This typically is done by the addition of sulfonate groups on aromatic rings

or on alkyl tethers built off the central dye structure. The high negative charge character of

the sulfonate groups creates like charge repulsion between dye molecules, thus dramatically

reducing their tendency to interact. The presence of three to four sulfonate groups on a probe

usually creates a very hydrophilic molecule with low nonspeciﬁ c binding character in biological

solutions and greater QY than the corresponding dye without sulfonates.

The following sections describe some of the most popular ﬂ uorescent probes for use in labe-

ling biomolecules. These ﬂ uorophores are available from a number of manufacturers in several

different forms, with a variety of reactive groups able to couple to speciﬁ c functional groups

on target molecules or with different substituents added to increase QY or hydrophilicity.

By far, the most often used ﬂ uorescent core structures are derivatives of cyanine, ﬂ uorescein,

rhodamine, and coumarin—probably in that order. However, new ﬂ uorescent molecules are

being developed and reported continually. For instance, there is growing interest in the use

of lanthanide chelates for time-resolved ﬂ uorescence (Section 9, this chapter) or quantum dot

nanocrystals (Section 10, this chapter) for highly stable ﬂ uorescence in multiplex detection

schemes. Careful study of each dye ’s individual properties can lead to the successful labeling of

a biomolecule for any application.

Suggested references on ﬂ uorescent spectroscopy and the use of ﬂ uorescent probes include

Lakowicz (1991, 1999); Bright (1988); Dewey (1991); McGown and Warner (1990); Ploem and

Tanke (1987); Darzynkiewicz and Crissman (1990); Haugland (1991); and Waggoner (1990).

1. Fluorescein Derivatives

Fluorescein and its derivatives represent one of the most popular of all ﬂ uorescent labeling

agents. Its ﬂ uorescent character is created by the presence of a multi-ring aromatic structure due

to the planer nature of its upper, fused three-ring system ( Figure 9.4 ). In its most elementary

form, the molecule has a molecular weight of about 332 Da, but most modiﬁ cation reagents

based on ﬂ uorescein are derivatives of this basic structure prepared through substitutions off

the No. 5 or 6 carbons of its lower ring. The derivatives provide reactivity toward particular

functional groups in biomolecules, allowing rapid labeling of proteins and nucleic acids.

Fluorescein has an effective excitation wavelength range of about 488–495 nm, closely

matching the photon emission for an argon laser (488 nm). Some ﬂ uorescein derivatives are

sold using the 488 reference in their name to reﬂ ect this excitation characteristic (i.e., DyLight

488 from Thermo Fisher and Alexa 488 from Invitrogen). The dye ’s emission spectrum occurs

between 518 and 525 nm, depending on the derivative chosen. Under ideal conditions, its QY

can be as high as 0.75, however its ﬂ uorescent intensity fades when it is dissolved in buffers,

exposed to light, or stored for extended periods (Kawamura Jr., 1977). In environments below

pH 7.0, ﬂ uorescein ’s QY is signiﬁ cantly quenched. In addition, when derivatives of ﬂ uorescein

are conjugated to proteins, the degree of ﬂ uorescent quenching can be as high as 50 percent,

especially for relatively hydrophobic forms of the dye. Fluorescein derivatives having multiple

sulfonates on them help to alleviate the quenching problem when labeling at higher density, but

it doesn ’t completely eliminate it. Even so, the ﬂ uorophore usually maintains excellent detecta-

bility in assay systems.

The following sections describe the most important ﬂ uorescein derivatives having reactive

groups commonly used to label biomolecules.

Amine-Reactive Fluorescein Derivatives

Two general forms of amine-reactive ﬂ uorescein probes are available. Both of them react under

alkaline conditions with primary amines in proteins and other molecules to form stable, highly

ﬂ uorescent derivatives.

Fluorescein Isothiocyanate

Fluorescein isothiocyanate (FITC) is one of the most popular ﬂ uorescent probes ever created.

An isothiocyanate derivative of ﬂ uorescein is synthesized by modiﬁ cation of its lower ring at

Figure 9.4 Fluorescein derivatives are produced through modiﬁ cation at the C-5 or C-6 positions on the

lower ring.

1. Fluorescein Derivatives 401

402 9. Fluorescent Probes

the 5- or 6-carbon positions. The two resulting isomers are nearly identical in their reactivity

and spectral properties, including excitation and emission wavelengths and intensities. Their

chemical differences, however, may affect the separation of modiﬁ ed proteins from excess rea-

gent or the analysis of tagged molecules by electrophoresis. For this reason, most manufactur-

ers purify the carbon-5 derivative as the FITC reagent of choice.

Isothiocyanates react with nucleophiles such as amines, sulfhydryls, and the phenolate ion

of tyrosine side chains (Podhradsky et al., 1979). The only stable product, however, is with pri-

mary amine groups, and so FITC is almost entirely selective for modifying - and N-terminal

amines in proteins (Jobbagy and Kiraly, 1966). The reaction involves attack of the nucleophile

on the central, electrophilic carbon of the isothiocyanate group ( Figure 9.5 ). The resulting elec-

tron shift creates a thiourea linkage between FITC and the protein with no leaving group.

FITC can be dissolved in DMF as a concentrated stock solution prior to its addition to an

aqueous reaction mixture. This may make aliquoting small quantities of the compound easier.

The reagent is water-soluble above pH 6. The isothiocyanate group is reasonably stable in

aqueous solution for short periods, but will degrade. FITC also can break down and loose

activity upon storage. It is best, therefore, to use fresh reagent for modiﬁ cation purposes.

Storage should be done under desiccated conditions, protected from light, and at  20 °C.

The ﬂ uorescent properties of FITC include an absorbance maximum at about 495 nm and

an emission wavelength of 520 nm. Fluorescent quenching of the molecule is possible. Under

Figure 9.5 FITC reacts with amine-containing compounds to produce an isothiourea linkage.

concentrated conditions, ﬂ uorescein-to-ﬂ uorescein interactions result in energy transfer

and self-quenching, which reduces the luminescence yield. This phenomenon can occur with

ﬂ uorescein-tagged molecules, as well. If derivatization of a protein is done at too high a level,

the resultant QY of the conjugate will be depressed. Typically, modiﬁ cations of proteins involve

adding no more than 8–10 ﬂ uorescein molecules per protein molecule, with a 4–5 substitution

level considered optimal.

FITC has been used in numerous applications involving ﬂ uorescence detection. Antibodies

or their fragments can be labeled to detect antigens in cells, tissues sections, blots, or on sur-

faces (Clausen, 1988). Tagging molecules with FITC also is useful in detecting proteins after

electrophoretic separations (Strottmann et al., 1983), for microsequencing analysis of proteins

and peptides (Muramoto et al., 1984), in analysis of molecules using capillary zone electro-

phoresis (Cheng and Dovichi, 1988), and in tracking and detecting molecules involved in vari-

ous bio-interactions (Burtnick, 1984; Friedman and Ball, 1989).

The level of ﬂ uorescein modiﬁ cation in a macromolecule can be determined by measuring its

absorbance at or near its characteristic excitation maximum ( 498 nm). The number of ﬂ uor-

ochrome molecules per molecule of protein is known as the F / P ratio. This value should be

measured for all derivatives prepared with ﬂ uorescent tags. The ratio is important in predicting

the behavior of antibodies labeled with FITC (Hebert et al., 1967; Beutner, 1971). Using the

known extinction coefﬁ cient of FITC in solution at pH 13 ( 

498nm

 8.1–8.5  10

; McKinney

et al., 1964; Jobbagy and Jobbagy, 1973) a determination of derivatization level can be made

after excess FITC is removed. At pH 7.8, the absorbance of protein-coupled FITC decreases by

8 percent (van Dalen and Haaijman, 1974).

A general protocol for the modiﬁ cation of proteins, particularly immunoglobulins, with

FITC is given below. Slight modiﬁ cations to the amount of reagent added to the reaction may

be done to optimize the F / P ratio.

Protocol

1. Prepare a protein solution in 0.1 M sodium carbonate, pH 9.0, at a concentration of at

least 2 mg/ml.

2. In a darkened lab, dissolve FITC (Thermo Fisher) in dry DMSO at a concentration of

1 mg/ml. Do not use old FITC, as breakdown of the isothiocyanate group over time may

decrease coupling efﬁ ciency. Protect from light by wrapping in aluminum foil or using

amber vials.

3. In a darkened lab, slowly add 50–100 l of FITC solution to each ml of protein solution

(at 2 mg/ml concentration). Gently mix the protein solution as the FITC is added.

4. React for at least 8 hours at 4 ° C in the dark.

5. The reaction may be quenched by the addition of ammonium chloride to a ﬁ nal concen-

tration of 50 mM. Some protocols also include at this point the addition of 0.1 percent

xylene cylanol and 5 percent glycerol as a photon absorber and protein stabilizer, respec-

tively. React for a further 2 hours to stop the reaction by blocking remaining isothiocy-

anate groups.

6. Purify the derivative by gel ﬁ ltration using a PBS buffer or another suitable buffer for the

particular protein being modiﬁ ed. The use of a desalting resin with low exclusion limits

work well. To obtain complete separation, the column size should be 15–20 times the

size of the applied sample. Fluorescent molecules often nonspeciﬁ cally stick to the gel ﬁ l-

tration support, so reuse of the column is not recommended.

1. Fluorescein Derivatives 403

404 9. Fluorescent Probes

NHS-Fluorescein and NHS-LC-Fluorescein

N-hydroxysuccinimide (NHS)-ﬂ uorescein is another amine-reactive ﬂ uorescent probe that con-

tains a carboxy-succinimidyl ester group off the No. 5 or 6 carbons on ﬂ uorescein ’s lower-ring

structure (Khanna and Ullman, 1980; Vigers et al., 1988; Brinkley, 1992). The 5- and 6-isomers

are virtually identical in their reactivity and ﬂ uorescent characteristics. Similar to FITC (above),

NHS-ﬂ uorescein can be used to label proteins and other macromolecules that contain primary

amine groups. This reagent is more stable than FITC, especially in storage. The NHS ester reac-

tion proceeds rapidly at slightly alkaline pH values, resulting in a stable, amide-linked deriva-

tive (Chapter 2, Section 1.4 and Chapter 4, Section 1; Figure 9.6 ).

The ﬂ uorescent properties of NHS-ﬂ uorescein are similar to FITC. The wavelength of maxi-

mal absorbance or excitation for the reagent is 491 nm and its emission maximum is 518 nm,

exhibiting a visual color of green (Sheehan and Hrapchak, 1980). Its molar extinction coef-

ﬁ cient at 491 nm in a pH 8.0 buffer environment is 66,000 M

 1

. Other components in

solution as well as the pH can change this value.

NHS-ﬂ uorescein is insoluble directly in aqueous solution and should be dissolved in organic

solvent prior to addition of a small aliquot to a buffered reaction medium. Concentrated

stock solutions may be prepared in DMSO or DMF. Such solutions are relatively stable if pro-

tected from light. Reaction conditions should be maintained at the optimal reactivity for NHS

esters—pH 7–9.

NHS-LC-ﬂ uorescein (Invitrogen) is an analog of NHS-ﬂ uorescein that contains a

6-aminocaproic acid spacer group, extending the NHS ester group away from the ﬂ uorescein

portion. The longer length of the coupling arm may decrease steric hindrance around the ﬂ uo-

rescent head of the molecule, thus reducing any ﬂ uorescence quenching due to its attachment

to a macromolecule. All other properties of the long-chain version are virtually identical to that

described above for NHS-ﬂ uorescein.

Sulfonated versions of NHS-ﬂ uorescein dyes are available that contain negative charges to

make the molecule more water-soluble and avoid nonspeciﬁ c interactions with biomolecules

(Thermo Fisher and Invitrogen). These 488-type dyes are a better choice than the original ﬂ u-

orescein dyes, because of their advantages when working with proteins and other biological

samples, including greater brightness, lower nonspeciﬁ c binding, and less tendency to quench.

The NHS ester reactions of the sulfonated dyes are identical to those of the non-sulfonated

types, but the sulfonated ones can be labeled on proteins at higher densities without ﬂ uores-

cence quenching or protein precipitation.

The following procedure is a suggested method for using NHS-ﬂ uorescein to label

immunoglobulins.

Protocol

1. Dissolve the IgG to be labeled in 50 mM sodium bicarbonate buffer, pH 8.5, at a concen-

tration of 10 mg/ml.

2. Dissolve 0.5 mg of NHS-ﬂ uorescein (Thermo Fisher) in 0.5 ml DMSO. Protect from

light.

3. In a darkened lab, slowly add 50–100 l of the NHS-ﬂ uorescein solution to the antibody

solution, while mixing. Protect from light by wrapping the reaction vessel in aluminum foil.

4. React for 2 hours on ice.

5. Remove unreacted NHS-ﬂ uorescein and reaction by-products by gel ﬁ ltration or dialysis.

Continue to protect all labeled protein solutions from light.

Figure 9.6 NHS-ﬂ uorescein reacts with amine-containing compounds via its NHS ester to form amide bonds.

1. Fluorescein Derivatives 405

406 9. Fluorescent Probes

A spectrophotometric assessment of the F / P ratio should be done after puriﬁ cation of the

tagged antibody. The measurement of absorbance at 495 nm (for ﬂ uorescein) divided by the

absorbance at 280 nm should be between 0.3 and 1.0 to obtain a good ﬂ uorescent derivative of

acceptable activity and low background. This usually translates into a ratio of about 4–7 ﬂ uo-

rescein molecules per protein molecule.

Sulfhydryl-Reactive Fluorescein Derivatives

Fluorescein derivatives containing a sulfhydryl-reactive group off the lower-ring structure are

available to direct the modiﬁ cation reaction to more limited sites on target molecules. Coupling

through sulfhydryls instead of amines can help to avoid active centers in proteins, thus preserv-

ing activity in the ﬂ uorescent probe complex. Sulfhydryl reaction sites can be naturally avail-

able through free cysteine side chains, generated by reduction of disulﬁ des, or created by the

use of thiolation reagents (Chapter 1, Section 4.1).

The ﬁ rst two compounds discussed in this section are truly sulfhydryl-reactive, using the

common iodoacetyl and maleimide functionalities, respectively. The third derivative, however,

is not reactive directly with sulfhydryl groups, but contains a protected sulfhydryl which, after

deprotection, can be used to react with other sulfhydryl-reactive crosslinkers.

5-(and 6)-iodoacetamidoﬂ uorescein

The iodoacetamido derivatives of ﬂ uorescein possess a sulfhydryl-reactive iodoacetyl group

(Chapter 1, Section 4.2 and Chapter 2, Section 2.1) at either the 5- or 6-carbon position on

their lower ring. The isomers are commercially available in puriﬁ ed form, since some reactivity

and speciﬁ city differences between the 5- and 6-derivatives toward various sulfhydryl sites in

proteins may be observed. Both iodoacetamido derivatives are among the most intense ﬂ uoro-

phores available for labeling biomolecules due to high QY.

The iodoacetyl group of both isomers reacts with sulfhydryls under slightly alkaline conditions

to yield stable thioether linkages ( Figure 9.7 ). They do not react with unreduced disulﬁ des in cystine

residues or with oxidized glutathione (Gorman et al., 1987). The thioether bonds will be hydro-

lyzed under conditions necessary for complete protein hydrolysis prior to amino acid analysis.

Figure 9.7 5-IAF can be used to modify sulfhydryl-containing molecules, creating stable thioether linkages.

Care must be taken to protect these reagents from light, not only to maintain the ﬂ uorescent

yield of the ﬂ uorescein probe, but also to protect the iodoacetyl group from light-catalyzed

breakdown. Iodoacetamidoﬂ uorescein is soluble in DMF and also in aqueous solutions main-

tained above pH 6.0. Concentrated stock solutions may be prepared in DMF prior to addition

of a small aliquot to a reaction mixture. Protect solutions from light by wrapping in aluminum

foil and working in subdued light.

The spectral properties of these derivatives are similar to native ﬂ uorescein. The excitation

maximum occurs at about 490–495 nm and its emission peak at 515–520 nm, producing light

in the green region of the spectrum. The extinction coefﬁ cient of 5-iodoacetamidoﬂ uorescein

at its wavelength of maximum absorbance, 491 nm, is 82,000 M

 1

(pH 9), whereas the

extinction coefﬁ cient of 6-iodoacetamidoﬂ uorescein is 77,000 M

 1

at 493 nm (pH 9).

The 5-iodoacetamido derivative of ﬂ uorescein (5-IAF) has been used to label numerous pro-

teins and other biomolecules, including actin (Plank and Ware, 1987), myosin (Aguirre et al .,

1986), troponin (Greene, 1986), hemoglobin (Hirsch et al., 1986), and sulfhydryl-containing

proteins separated by SDS electrophoresis (Gorman, 1984).

The 6-iodoacetamido derivative (6-IAF) has been used to label myosin (Ando, 1984), actin

(Konno and Morales, 1985), microtubule-associated proteins (Scherson et al., 1984), and his-

tones (Cocco et al ., 1986).

The following protocol for labeling proteins with 5-IAF is adapted from Gorman (1987). It

is a bit unusual in that it involves reduction of disulﬁ des with dithiothreitol (DTT) and imme-

diate reaction with 5-IAF in excess without removal of excess reductant. The procedure can be

changed to include a gel ﬁ ltration step after disulﬁ de reduction to remove excess DTT, but in

any case, it should be optimized for each protein to be modiﬁ ed. An alternative to the use of

DTT to produce sulfhydryls is thiolation with a compound that can generate free thiols upon

reaction with a protein (Chapter 1, Section 4.1).

Protocol

1. Dissolve a protein-containing disulﬁ de residues at a concentration of 5–10 mg/ml in

0.1 M ammonium carbonate containing 1 percent SDS and 20 mM DTT. Note: The pres-

ence of detergent may be eliminated if certain disulﬁ des can be reduced in the protein

1. Fluorescein Derivatives 407

408 9. Fluorescent Probes

without completely denaturing it, such as in the reduction of antibodies in the hinge

region. If only partial reduction is done, then the amount of DTT should be reduced

to about a 3-fold molar excess over the concentration of antibody present (Sun et al. ,

2005). For this type of labeling the reaction buffer should be 50 mM sodium borate,

pH 8.5.

2. For complete reduction of all disulﬁ des in the presence of a denaturant, react for 16

hours at 0 °C and 2 hours at room temperature. For partial reduction of disulﬁ des, the

reaction time may be reduced to 2 hours at 37 °C, particularly for antibody thiol reduc-

tion, if only partial reduction of thiols in the hinge region is done.

3. Add a 5-fold molar excess of 5-IAF (Thermo Fisher) over the amount of DTT present.

The ﬂ uorescent probe may be solubilized in DMF prior to addition of a small aliquot to

the reaction mixture. Do not exceed 10 percent DMF in the ﬁ nal aqueous solution.

4. React for 2 hours at room temperature in the dark.

5. To recover a protein that has been completely reduced and labeled, precipitate the pro-

tein by the addition of 9 volumes of methanol at  20 °C. Collect the protein pellet by

centrifugation at 8,000 g for 15 minutes (4 °C). If partial reduction was done followed

by labeling with 5-IAF, then purify the labeled protein by gel ﬁ ltration or dialysis using a

molecular weight cutoff of about 5,000 D.

An alternative protocol for labeling sulfhydryl-containing proteins that does not require

DTT reduction can be found in a method adapted from Ando (1984). When preparing any

ﬂ uorescently labeled protein, optimization of the dye-to-protein ratio is important to obtain

the best performance in the intended application.

Protocol

1. Prepare a 20 mM 6-IAF (Thermo Fisher) solution by dissolving 10.3 mg/ml of DMF.

Prepare fresh and protect from light.

2. Dissolve the protein to be modiﬁ ed at a concentration of 5–10 mg/ml in 20 mM TES, pH

7.0. TES is 2-[[tris(hydroxymethyl)methyl]amino]ethanesulfonic acid.

3. Slowly add 25–50 l of the 6-IAF solution to each ml of the protein solution while mixing.

4. React for 2 hours at 4 °C in the dark.

5. Remove excess reactant and reaction by-products by gel ﬁ ltration using a desalting resin

or dialysis.

Fluorescein-5-maleimide

Fluorescein-5-maleimide is a ﬂ uorescent probe containing a sulfhydryl-reactive maleimide

group on its lower-ring structure. Modiﬁ cation of sulfhydryl-containing molecules under physi-

ological pH conditions results in stable thioether bonds (Chapter 2, Section 2.2) (

Figure 9.8 ).

The derivative thus possesses ﬂ uorescent properties closely characteristic of ﬂ uorescein mol-

ecules: excitation wavelength  490 nm; emission wavelength  515 nm, in the green spectral

region. Conjugates prepared by ﬂ uorescein-5-maleimide are among the most intensely ﬂ uorescent

probes available. The reactivity of the maleimide group is similar to that of the iodoacetyl

derivative discussed previously.

Thus, this reagent can be used to label ﬂ uorescently proteins and other biomolecules con-

taining free sulfhydryl residues. If there are no  SH groups available, their creation can be

accomplished by reduction of indigenous disulﬁ des or through the use of various thiolation

reagents (Chapter 1, Section 4.1).

Fluorescein-5-maleimide is slightly soluble in aqueous solutions above pH 6 ( 1 mM concen-

tration). It may be dissolved in DMF at higher concentrations and a small addition of this solu-

tion made to an aqueous reaction mixture to initiate labeling. Do not exceed 10 percent DMF

in the reaction buffer to avoid protein precipitation. At pH 8, the reagent has an extinction

coefﬁ cient at 490 nm of about 78,000 M

 1

Fluorescein-5-maleimide has been used in numerous applications, including labeling the

transmembrane glycoprotein H-2K

on both the N- and C-terminal regions to investigate the

structure of the molecule (Cardoza et al., 1984), for the determination of two different confor-

mations of the protein actin (Konno and Morales, 1985), in the study of a bacterial sensory

receptors (Falke et al., 1988), in the structural mapping of chloroplast coupling factor (Snyder

and Hammes, 1984, 1985), for localization of the stilbenedisulfonate receptor on human

Figure 9.8 Fluorescein-5-maleimide can be used to modify sulfhydryl groups, forming thioether bonds.

1. Fluorescein Derivatives 409