Hermanson G. Bioconjugate Techniques, Second Edition

Подождите немного. Документ загружается.

410 9. Fluorescent Probes

erythrocytes (Rao et al., 1979), investigating the calcium-dependent ATPase protein structure

of sarcoplasmic reticulum (Bigelow and Inesi, 1991), and to study the movement of tRNA dur-

ing peptide bond formation on ribosomes (Odom et al ., 1990).

Fluorescein-5-maleimide also has been used to study the assembly dynamics of mycobacte-

rium tuberculosis (Chen et al., 2007), to study monomers and dimers of nhaa Na



anti-

porter of E. coli (Rimon et al., 2007), and to investigate the regulation of the protein disulﬁ de

proteome by mitochondria (Yang et al. , 2007).

Protocol

1. Dissolve a sulfhydryl-containing protein or other macromolecule at a concentration of

1–10 mg/ml in 20 mM sodium phosphate, 0.15 M NaCl, pH 7.2. Other buffers within the

range of pH 6.5–7.5 may be used as long as they don ’t contain extraneous sulfhydryls.

2. Dissolve ﬂ uorescein-5-maleimide (Thermo Fisher, Invitrogen) in DMF at a concentration

of 10 mM (4.25 mg/ml).Protect from light.

3. In subdued lighting conditions, add 25–50 l of the ﬂ uorescein solution to each ml of

protein solution while mixing. Alternatively, determine the exact molar quantity of pro-

tein present and add a 25-fold molar excess of ﬂ uorescein-5-maleimide solution.

4. React for 2–4 hours at room temperature in the dark. The reaction also may be done at

0–4°C, but allow at least 8 hours for completion.

5. Immediately purify the derivative using gel ﬁ ltration on a desalting resin. Protect the

solutions from light during the chromatography.

SAMSA-Fluorescein

SAMSA-ﬂ uorescein, 5-{[2(and 3)-5-(acetylmercapto)-succinoyl]amino}ﬂ uorescein, is a ﬂ uores-

cent probe containing a protected sulfhydryl group. In its protected state, the compound is unre-

active. The acetyl-protecting group can be removed by treatment with dilute NaOH at pH 10.0

(Figure 9.9 ). The resulting free sulfhydryl derivative can be used to label thiol-reactive cross-

linkers or to couple with sulfhydryl residues on proteins and other molecules. After activating

Figure 9.9 SAMSA-ﬂ uorescein contains a protect thiol that can be deblocked by treatment with hydroxy-

lamine. The reagent then can be used to modify molecules containing sulfhydryl-reactive groups.

proteins with crosslinkers containing terminal maleimide, pyridyl disulﬁ de, or iodoacetyl

groups, SAMSA-ﬂ uorescein can be used to assess the level of modiﬁ cation. For instance, a male-

imide-activated protein that has been derivatized with succinimidyl-4-( N -maleimidomethyl)

cyclohexane-1-carboxylate (SMCC) could be reacted with this reagent to yield a ﬂ uorescein

derivative that can be assayed spectroﬂ uorometrically for its level of ﬂ uorescence. Using the

molar extinction coefﬁ cient for SAMSA-ﬂ uorescein (  at 495 nm  80,000 M

 1

), the

molar level of incorporation of the label can be calculated. This determination directly corre-

lates to the original level of maleimide groups present on the protein.

SAMSA-ﬂ uorescein is an orange solid compound. Dissolved in buffer at pH 9.0, its maximal

wavelength of absorption or excitation is at 495 nm, while its emission wavelength maximum

is 520 nm. The reagent and all solutions and derivatives made from it are light sensitive and

should be stored in the dark. SAMSA-ﬂ uorescein is soluble in aqueous solutions above pH 6.0,

but it can be dissolved in DMF to prepare a concentrated stock solution prior to adding a small

amount to a buffered reaction mixture.

Protocol

1. To deprotect the acetylated sulfhydryl, dissolve the desired amount of SAMSA-ﬂ uorescein

(Invitrogen) in 100 mM NaOH, pH 10.0, at a concentration of 10 mg/ml.

2. React for 15 minutes at room temperature.

3. Lower the pH to 7–8 by the addition of solid sodium phosphate.

4. Add the required amount of deprotected SH-ﬂ uorescein to a protein or other macromol-

ecule that had been modiﬁ ed to contain a sulfhydryl-reactive group. Use a 5- to 10-fold

molar excess of SH-ﬂ uorescein to the expected amount of sulfhydryl-reactivity present.

5. React for 2 hours at room temperature, protected from light.

6. Remove excess ﬂ uorescent probe by gel ﬁ ltration using a desalting resin.

7. Measure the absorbance of the derivative at 495 nm. Determine the level of ﬂ uorophore

incorporation by using its molar extinction coefﬁ cient.

1. Fluorescein Derivatives 411

412 9. Fluorescent Probes

Aldehyde/Ketone and Cytosine-Reactive Fluorescein Derivatives

Hydrazide groups directly react with aldehyde and ketone groups to form relatively stable

hydrazone linkages (Chapter 2, Section 5.1). Two ﬂ uorescein derivatives are commonly avail-

able that contain hydrazide groups off their No. 5 carbons on the lower-ring structure. Both

may be used to label ﬂ uorescently aldehyde- or ketone-containing molecules. Although most

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

oproteins, RNA, and other molecules containing sugar residues can be oxidized with 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 a hydrazide-reactive site

(Chapter 1, Section 4.4).

DNA and RNA may be modiﬁ ed with hydrazide-reactive probes by reacting their cyto-

sine residues with bisulﬁ te to form reactive sulfone intermediates. These derivatives undergo

transamination to couple hydrazide- or amine-containing probes (Draper and Gold, 1980)

(Chapter 27, Section 2.1).

Fluorescein-5-thiosemicarbazide

Fluorescein-5-thiosemicarbazide is a hydrazide derivative of ﬂ uorescein that can spontaneously

react with aldehyde- or ketone-containing molecules to form a covalent, hydrazone linkage

(Figure 9.10 ) (Invitrogen). It also can be used to label cytosine residues in DNA or RNA by

use of the bisulﬁ te activation procedure (Chapter 27, Section 2.1). The resulting ﬂ uorescent

derivative exhibits an excitation maximum at a wavelength of 492 nm and a maximal emission

wavelength of 519 nm when dissolved in buffer at pH 8.6. In the same buffered environment,

the compound has an extinction coefﬁ cient of approximately 78,000 M

 1

at 492 nm.

Fluorescein-5-thiosemicarbazide is soluble in DMF or in buffered aqueous solutions at pH

values above 7.0. The reagent may be dissolved in DMF as a concentrated stock solution before

adding a small aliquot to an aqueous reaction medium. The compound itself and all solutions

made with it should be protected from light to avoid decomposition of its ﬂ uorescent properties.

This hydrazide derivative of ﬂ uorescein has been used in a number of applications, includ-

ing site-directed labeling of antibodies through their carbohydrate chains (Duijndam et al .,

1988), labeling thrombin and anti-thrombin (Atha et al., 1964), the Na



-ATPase glycopro-

tein (Lee and Fortes, 1985), periodate-oxidized RNA (Odom et al., 1980, 1984; Ferguson and

Yang, 1986; Friedrich et al., 1988), for the determination of carbonyl groups in proteins and

to detect oxidized glycoproteins in gels (Ahn et al., 1987). It also has been used to investigate

the molecular basis of RNA recognition (Pagano et al., 2007), in a non-invasive visualization

method for assessment of carbonylated protein (Fujita et al., 2007), and for the detection of

protein carbonyls in aging liver tissue (Chaudhuri et al. , 2007).

The following protocols are generalized for the labeling of cell-surface glycoproteins or glyc-

oproteins in solution. Some optimization may be necessary to achieve the best level of ﬂ uores-

cent modiﬁ cation for each particular application.

Protocol for Labeling Cell Surfaces

1. Add 10

–10

cells/ml in a PBS solution (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4)

containing 1 mM sodium periodate and incubate on ice for 30 minutes in the dark. This

level of periodate addition will target the oxidation only to sialic acid residues (Chapter 1,

Section 4.4). If additional sites of glycosylation also are to be labeled, increase the perio-

date concentration to 10 mM and do the reaction at room temperature in the dark.

2. Centrifuge and wash cells several times with PBS. Some protocols include a quench

step wherein excess sodium periodate is eliminated by addition of glycerol. This can be

accomplished by adding 3 volumes of 0.1 M glycerol in PBS prior to centrifugation.

3. Resuspend cells in PBS containing 0.5 mg/ml ﬂ uorescein-5-thiosemicarbazide.

4. Incubate 30 minutes in the dark at room temperature.

5. To reduce the hydrazone bonds to more stable linkages, cool the cell suspension to 0 °C and

add an equal volume of 30 mM sodium cyanoborohydride in PBS. Incubate for 40 minutes.

Note: If the presence of a reducing agent is detrimental to protein activity, eliminate the

reduction step. In most cases, the hydrazone linkage is stable enough for ﬂ uorescent labe-

ling experiments.

6. Centrifuge and wash cells extensively with PBS.

Figure 9.10 Fluorescein-5-thiosemicarbazide reacts with aldehyde groups to produce hydrazone linkages.

1. Fluorescein Derivatives 413

414 9. Fluorescent Probes

Protocol for Labeling Glycoproteins in Solution

1. Dissolve the glycoprotein(s) to be labeled in ice-cold 1 mM sodium periodate, 10 mM

sodium phosphate, 0.15 M NaCl, pH 7.4, for the exclusive oxidation of sialic acid resi-

dues. For general carbohydrate oxidation, increase the periodate concentration to 10 mM

in PBS at room temperature.

2. React for 30 minutes on ice (for sialic acids) or at room temperature (for other polysac-

charide residues).

3. Remove excess reactant by gel ﬁ ltration using a desalting resin with PBS, pH 7.4. Some

protocols use a quenching agent to remove excess periodate prior to gel ﬁ ltration. This

can be done by adding glycerol to a ﬁ nal concentration of 0.1 M.

4. To the puriﬁ ed, oxidized glycoprotein(s), add ﬂ uorescein-5-thiosemicarbazide to a ﬁ nal

concentration of 0.5 mg/ml.

5. React for 30 minutes at room temperature in the dark.

6. To reduce the hydrazone bonds to more stable linkages, cool the solution to 0 °C and add

an equal volume of 30 mM sodium cyanoborohydride in PBS. Incubate for 40 minutes.

Note: If the presence of a reducing agent is detrimental to protein activity, eliminate the

reducing step. In most cases, the hydrazone linkage is stable enough for ﬂ uorescent labe-

ling experiments.

7. Purify the ﬂ uorescently labeled glycoprotein(s) by gel ﬁ ltration using a desalting resin.

5-(((2-(carbohydrazino)methyl)thio)acetyl)-aminoﬂ uorescein

Another hydrazine derivative of ﬂ uorescein, 5-(((2-(carbohydrazino)methyl)thio)acetyl)-

aminoﬂ uorescein, contains a longer spacer arm off its No. 5 carbon atom of its lower ring than

ﬂ uorescein-5-thiosemicarbazide, described previously (Invitrogen). The reagent can be used to

react spontaneously with aldehyde- or ketone-containing molecules forming a hydrazone link-

age ( Figure 9.11 ). It also can be used to label cytosine residues in DNA or RNA by use of

the bisulﬁ te activation procedure (Chapter 27, Section 2.1). The resulting ﬂ uorescent derivative

exhibits a maximal excitation at 490 nm and a maximal luminescence emission peak at 516 nm

when dissolved in buffer at pH 8.0. In the same buffered environment, the compound has an

extinction coefﬁ cient of approximately 75,000 M

 1

at 490 nm.

The ﬂ uorescent probe, 5-(((2-(carbohydrazino)methyl)thio)acetyl)-aminoﬂ uorescein, is solu-

ble in DMF or in buffered aqueous solutions at pH values above 7.0. The reagent may be dis-

solved in DMF as a concentrated stock solution before adding a small aliquot to an aqueous

reaction medium. The compound itself and all solutions made with it should be protected from

light to avoid decomposition of its ﬂ uorescent properties.

The methods for using this reagent in labeling glycoproteins on cell surfaces or in solution

are similar to those described for ﬂ uorescein-5-thiosemicarbazide, above.

2. Rhodamine Derivatives

Rhodamine and its derivatives are popular ﬂ uorescent probes for labeling all types of biomol-

ecules. Their ﬂ uorescent character is created by the presence of a planer, multi-ring aromatic

structure similar to ﬂ uorescein, but with nitrogen atoms replacing the oxygens on the outer

rings ( Figure 9.12 ). Fluorescent modiﬁ cation reagents based on rhodamine are derivatives of

this basic structure. Activated rhodamine probes have reactive groups prepared through substi-

tutions off the No. 5 or 6 carbons of its lower ring. These derivatives provide reactivity toward

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

acids. Other alterations to the basic rhodamine structure modulate its ﬂ uorescent character,

creating more intense or stable ﬂ uorophores, or changing its wavelength of excitation and

emission toward the red region. Many such derivatives are now commercially available.

The tetramethylrhodamine derivative, for instance, has two methyl groups attached to each

nitrogen on its outer rings. Activated forms of tetramethylrhodamine are among the most com-

mon derivatives of rhodamine used for ﬂ uorescent labeling. Another useful derivative is rhod-

amine B, which contains two ethyl groups on each nitrogen as well as a carboxylate group at

the No. 3 position on its lower ring. Rhodamine 6G adds two methyl groups on the outer rings

as well as an ethyl ester group off rhodamine B ’s carboxylate. Rhodamine 110 contains no

Figure 9.11 This carbohydrazide-containing ﬂ uorescein derivative can be used to modify aldehyde-containing mole-

cules. Glycoconjugates may be labeled with this reagent after treatment with sodium periodate to produce aldehydes.

2. Rhodamine Derivatives 415

416 9. Fluorescent Probes

substituents on the upper nitrogens and only the carboxylate on the lower ring. Sulforhodamine

B possesses rhodamine B ’s two ethyl groups on each nitrogen of the upper rings, but has two sul-

fonates at the No. 3 and 5 positions of its lower ring. This derivative is often called Lissamine ™

rhodamine B—Lissamine being a trademark of Imperial Chemical Industries. Another popular

derivative of rhodamine, sulforhodamine 101, goes by the name of Texas Red ™ (a trademark

of Invitrogen). This derivative has intense luminescent properties that take it the farthest into

the red region of the spectrum. The basic structures of these rhodamine derivatives are shown in

Figure 9.13 . The corresponding commercially available rhodamine ﬂ uorophores usually contain

additional reactive groups, on the No. 5 or 6 carbons of the lower ring, to permit coupling to

target molecules (Invitrogen, Thermo Fisher).

Rhodamine derivatives have effective excitation wavelengths within the visible light spec-

trum from the low- to high-500 nm range, depending on the particular derivative. Their asso-

ciated emission wavelengths occur from the mid- to high-500 nm range—with Texas Red

derivatives typically emitting at over 600 nm–within the orange-to-red visible spectrum. The

QY of rhodamine derivatives is generally less than that of ﬂ uorescein–only about 25 percent.

However, its ﬂ uorescent intensity fades more slowly than ﬂ uorescein when it is dissolved in

buffers, exposed to light, or stored for extended periods. In addition, its orange-to-red lumi-

nescence is in stark contrast to the green of ﬂ uorescein, thus these two types of probes form an

ideal pair for use in double-staining techniques, especially in ﬂ uorescent microscopy.

The following sections describe the most important rhodamine derivatives commonly used

to label biomolecules.

Amine-Reactive Rhodamine Derivatives

Four forms of amine-reactive rhodamine probes are commonly available. Two of them are

based on the tetramethyl derivatives of the fundamental rhodamine structure, one is based on

the sulforhodamine B or Lissamine derivative, and the last is the sulforhodamine 101 or Texas

Red-type of derivative. All of them react under alkaline conditions with primary amines in pro-

teins and other molecules to form stable, highly ﬂ uorescent complexes.

Tetramethylrhodamine-5-(and 6)-isothiocyanate

Tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC) is one of the most popular ﬂ uores-

cent probes available. The isothiocyanate derivative of tetramethylrhodamine is synthesized by

Figure 9.12 The basic structure of rhodamine derivatives.

modiﬁ cation of its lower ring at the 5- or 6-carbon positions. The two resulting isomers are

almost identical in their reactivity but slightly different in their spectral properties, including

excitation and emission wavelengths and intensities. The chemical differences in the isomers,

however, may affect the separation of modiﬁ ed proteins from excess probe or the analysis of

tagged molecules by electrophoresis. For this reason, most manufacturers offer the mixed iso-

mers as well as the puriﬁ ed 5- or 6-isothiocyanate derivatives individually.

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 TRITC is almost entirely selective for modifying - and N-terminal

amines in proteins. The reaction involves attack of the nucleophile on the central, electrophilic

Figure 9.13 The primary rhodamine derivatives useful for ﬂ uorescent labeling.

2. Rhodamine Derivatives 417

418 9. Fluorescent Probes

carbon of the isothiocyanate group ( Figure 9.14 ). The resulting electron shift creates a thiourea

linkage between TRITC and the protein with no leaving group.

TRITC is relatively insoluble in water, but it can be dissolved in DMF or DMSO as a concen-

trated stock solution prior to its addition to an aqueous reaction mixture. The isothiocyanate

group is reasonably stable in aqueous solution for short periods, but will degrade by hydrolysis.

TRITC also is more stable to photobleaching than FITC (Section 1, this chapter), and its

absorption and emission spectra are less sensitive to environmental conditions, such as pH.

It is best, however, to use only 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 TRITC (mixed isomers) include an absorbance maximum at

about 544 nm and an emission wavelength of 570 nm. Fluorescent quenching of the molecule

is possible. Under concentrated conditions, rhodamine-to-rhodamine interactions result in self-

quenching which reduces its luminescence yield. This phenomenon can occur with TRITC-

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 from expected values. Typically, modiﬁ cations of pro-

teins involve adding no more than 8–10 rhodamine molecules per molecule of protein, with a

4–5 substitution level considered optimal.

TRITC has been used in numerous applications involving ﬂ uorescence detection, including

double-staining techniques with ﬂ uorescein-labeled probes (Mossberg and Ericsson, 1990), the

synthesis of ﬂ uorescently labeled DNA probes (Smith et al., 1985), as a label in homogeneous

Figure 9.14 TRITC reacts with amine-containing molecules to create an isothiourea linkage.

immunoassay systems (Nithipatikom and McGown, 1987), to investigate speciﬁ c interactions of

proteins with cell surfaces (Hochman et al., 1988), and as an important ﬂ uorescent tag of anti-

bodies in immunohistochemical staining techniques (Davidson and Hilchenbach, 1990). The

ﬂ uorescent dye also has been used to investigate dynein-dependent transport of virus proteins

(Ramanathan et al., 2007), the trafﬁ cking of the prion protein (Campana et al., 2007), and the dis-

tribution of FAT1 isoforms in migrating cells (Braun et al., 2007). Many thousands of additional

references cite the use of TRITC in labeling molecules in ﬂ uorescence detection applications.

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

absorbance at or near its characteristic absorption maximum ( 575 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 especially important

in predicting the behavior of antibodies labeled with TRITC. For a TRITC-labeled protein, the

ratio of its absorbance at 575–280 nm should be between 0.3 and 0.7.

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

TRITC is given below. 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 TRITC (Thermo Fisher) in dry DMSO at a concentration of

1 mg/ml. Do not use old TRITC, 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 l of TRITC solution to each ml of protein solution.

Gently mix the protein solution as the TRITC is added.

4. React for at least 8 hours at 4 ° C in the dark or 2–4 hours at room temperature.

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

tion 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, respectively. React

for a further 2 hours to stop the reaction by blocking remaining isothiocyanate 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 (e.g., Excellulose, Thermo

Fisher) or similar matrices with low exclusion limits work well. To obtain complete sepa-

ration, the column size should be 15–20 times the size of the applied sample. Fluorescent

molecules often nonspeciﬁ cally stick to gel ﬁ ltration supports, so reuse of the column is

not recommended.

NHS-Rhodamine

NHS-rhodamine is an amine-reactive ﬂ uorescent probe that contains a carboxy-succinimidyl

ester group off the No. 5 or 6 carbons on rhodamine ’s lower-ring structure (Kellogg et al.,

1988). The 5- and 6-isomers are virtually identical in their reactivity and ﬂ uorescent characteris-

tics. Similar to TRITC (described previously), NHS-rhodamine can be used to label proteins and

other macromolecules that contain primary amine groups. The isomeric forms of the ﬂ uorescent

probe are available in mixed and puriﬁ ed forms (Invitrogen, Thermo Fisher). The pure forms are

2. Rhodamine Derivatives 419