Hermanson G. Bioconjugate Techniques, Second Edition

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420 9. Fluorescent Probes

particularly important for labeling nucleic acid probes that will be separated by electrophoresis

(Chehab and Kan, 1989). The NHS ester labeling reaction proceeds rapidly at slightly alkaline

pH values, resulting in a stable, amide-linked derivative (Chapter 2, Section 1.4; Figure 9.15 ).

The ﬂ uorescent properties of NHS-rhodamine are similar to TRITC. The wavelength of

maximal absorbance or excitation for the reagent is 544 nm and its emission maximum is

576 nm, exhibiting a visual color of orange-red. Its molar extinction coefﬁ cient at 546 nm in

a methanol environment is 63,000 M

 1

. Other components in solution as well as the pH

(in aqueous buffers) can change this value.

NHS-rhodamine 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 for short

Figure 9.15 NHS-rhodamine can be used to label amine-containing molecules via its NHS ester group.

periods if protected from light, but should be prepared fresh. Reaction conditions should be

maintained at the optimal reactivity for NHS esters, which is pH 7–9.

NHS-rhodamine has been used in numerous applications, including the detection of spe-

ciﬁ c DNA sequences (Chehab and Kan, 1989), studying the behavior of microtubules and actin

ﬁ laments in living Drosophila embryos (Kellogg et al., 1988), investigation into the light-initi-

ated breakup of microtubules (Vigers et al., 1988), studying -conotoxin-sensitive channels in

neurons (Jones et al., 1989), investigating growth cones during axon elongation (Tanaka and

Kirschner, 1991), and in studying the pathways of mitotic spindle assembly in vitro (Sawin and

Mitchison, 1991). NHS-rhodamine also has been used to perform ﬂ uorescent studies of mem-

brane protein complexes (Wittig et al., 2007), to investigate the cross-bridge between microtu-

bules (Li et al. , 2007), and study a multi-enzyme network in digestion (Delcroix et al. , 2006).

The following generalized protocol relates to the labeling of IgG with NHS-rhodamine.

Optimization of the level of rhodamine incorporation may have to be done with other proteins

or other macromolecules.

Protocol

1. Dissolve an immunoglobulin to be labeled in ice-cold, 50 mM sodium bicarbonate, pH

8.5, at a concentration of 10 mg/ml.

2. Dissolve NHS-rhodamine at a concentration of 1 mg/ml in DMSO. Protect from light.

3. In a darkened lab, slowly add 50–100 l of the NHS-rhodamine solution to each ml of

the antibody solution with mixing. Wrap the vessel with aluminum foil to protect from

light.

4. Place the sample on ice and react for 2 hours.

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

Lissamine Rhodamine B Sulfonyl Chloride

The Lissamine form of rhodamine B consists of diethyl modiﬁ cations on the two nitrogens of

the upper rings of the basic rhodamine molecule as well as two sulfonate groups added at the

2. Rhodamine Derivatives 421

422 9. Fluorescent Probes

3- and 5-carbon positions of the lower ring. Lissamine rhodamine B sulfonyl chloride is an

amine-reactive reagent made by converting the No. 5 sulfonate group to a reactive sulfonyl

halide. Reaction with proteins and other amine-containing molecules results in the formation

of sulfonamide bonds ( Figure 9.16 ). Lissamine is a trademark of Imperial Chemical Industries.

The spectral characteristics of protein conjugates made with Lissamine rhodamine B deriva-

tives are of longer wavelength than those of tetramethylrhodamine—more toward the red region

of the spectrum. In addition, modiﬁ ed proteins have better chemical stability and are somewhat

easier to purify than those made from TRITC (discussed previously). Lissamine derivatives also

make more photostable probes than the ﬂ uorescein derivatives (Section 1, this chapter).

Lissamine rhodamine B sulfonyl chloride is relatively insoluble in water, but may be dis-

solved in DMF prior to the addition of a small aliquot to an aqueous reaction. Do not dissolve

in DMSO, as sulfonyl chlorides will readily react with this solvent (Boyle, 1966). The com-

pound has a maximal absorptivity at 556 nm with an extremely high extinction coefﬁ cient of

up to 93,000 M

 1

(in methanol) in highly puriﬁ ed form. Its emission maximum occurs at

576 nm, emitting red luminescence.

A sulfonyl chloride rapidly reacts with amines on proteins and other molecules to form sta-

ble sulfonamide bonds. It also may react with tyrosine  OH groups, aliphatic alcohols, thi-

ols, and imidazole groups (such as histidine side chains). Conjugates of sulfonyl chlorides with

sulfhydryls and imidazole rings are unstable, while esters formed with alcohols are subject to

nucleophilic displacement (Nillson and Mosbach, 1984; Scouten and Van der Tweel, 1984).

The only stable derivative with proteins therefore is the sulfonamide, formed by reaction with

-lysine and N-terminal amines. Optimal conditions for coupling are non-amine-containing

buffers in the pH range of 9–10. Phosphate, bicarbonate, or borate buffers are recommended

for the modiﬁ cation reaction. Avoid the presence of other nucleophiles that can cross-react with

the sulfonyl chloride (e.g., amine-containing components or sulfhydryl-containing reducing

agents). In aqueous solutions, hydrolysis is a competing reaction, but occurs more slowly with

sulfonyl halides than with the corresponding acid chlorides of carboxylate groups. Unreacted,

hydrolyzed probe is the water-soluble sulforhodamine B ﬂ uorophore which is easily removed

by gel ﬁ ltration or dialysis.

Lissamine rhodamine B sulfonyl chloride has been used in numerous applications, includ-

ing multiple-labeling techniques in microscopy (Wessendorf, 1990), for confocal microscopy

Figure 9.16 Lissamine rhodamine B sulfonyl chloride reacts with amine-containing molecules to produce stable

sulfonamide bonds.

techniques (Tsien and Waggoner, 1990), in the study of ﬁ bronectin receptors (Duband et al .,

1988), for investigations into microtubule and intermediate ﬁ lament association (Geiger and

Singer, 1980), for the labeling of glycoconjugates (Wilchek et al., 1980), for studying regula-

tion of the Na



-ATPase system (Sipe et al., 1991), and for investigations into the redox

potential within mitochondria (Chazotte and Hackenbrock, 1991). The dye also has been used

to study the shape of giant unilamellar vesicles as model plasma membranes (Gudheti et al. ,

2007), to investigate the activity of xyloglucan xylogucosyl transferase (Hrmova et al., 2007),

and the switching of neuroﬁ laments between mobile and stationary states (Trivedi et al., 2007).

Hundreds of additional biological studies have used this ﬂ uorescent probe.

The following protocol is a general guide for labeling biological macromolecules with

Lissamine rhodamine B sulfonyl chloride. Optimization of the ﬂ uorophore incorporation level

(F / P ratio) may have to be done for speciﬁ c labeling experiments.

Protocol

1. Dissolve the amine-containing macromolecule to be labeled (i.e., a protein) in 0.1 M

sodium carbonate/bicarbonate buffer, pH 9.0, at a concentration of 1–5 mg/ml.

2. Dissolve Lissamine rhodamine B sulfonyl chloride (Invitrogen) in DMF at a concentra-

tion of 1–2 mg/ml. Protect from light and use immediately.

3. In a darkened lab and with gentle mixing, slowly add 50–100 l of the ﬂ uorophore solu-

tion to the protein solution.

4. React for 1 hour at room temperature in the dark.

5. Remove excess ﬂ uorophore by gel ﬁ ltration using a desalting resin or by dialysis.

Texas Red Sulfonyl Chloride

Texas Red sulfonyl chloride is the active halogen derivative of sulforhodamine 101. This

important derivative of the basic rhodamine molecule possesses dual aliphatic rings off the

upper-ring nitrogens and sulfonate groups on the No. 3 and 5 carbon atoms of its lower-ring

component. The sulfonyl chloride group can react with primary amines in proteins and other

molecules to form stable sulfonamide bonds ( Figure 9.17 ). The group, however, can hydrolyze

2. Rhodamine Derivatives 423

424 9. Fluorescent Probes

in the presence of moisture. For this reason, only fresh Texas Red sulfonyl chloride should be

used for modiﬁ cation experiments.

The intense Texas Red ﬂ uorophore has a QY that is inherently higher than the tetrameth-

ylrhodamine or Lissamine rhodamine B derivatives. Texas Red ’s luminescence is shifted maxi-

mally into the red region of the spectrum, and its emission peak only minimally overlaps with

that of ﬂ uorescein. This makes Texas Red derivatives among the best choices of labels for use

in double-staining techniques.

Texas Red sulfonyl chloride has a maximal excitation at 589 nm and a maximum emission

at 615 nm when dissolved in methanol. The extinction coefﬁ cient of the compound dissolved

in acetonitrile is 85,000 M

 1

at 596 nm. The only disadvantage of this ﬂ uorophore is its

poor excitation by the standard argon laser at 488 nm. However, since both Texas Red and

ﬂ uorescein are weakly excited by an argon laser at 514 nm, it makes them fairly good pairs for

use in laser confocal microscopy or ﬂ ow cytometry (Mossberg and Ericsson, 1990). The ﬂ uor-

ophore is particularly appropriate for excitation by the 568 nm line produced by an argon–

krypton mixed laser used on some confocal devices. Compared to other rhodamine derivatives,

Texas Red ﬂ uorophores display low background in staining techniques and are among the

most photostable probes available.

Texas Red sulfonyl chloride is soluble in DMF or acetonitrile and may be dissolved as a con-

centrated stock solution in either solvent prior to the addition of a small aliquot to an aqueous

reaction medium. Avoid the use of DMSO, as sulfonyl chlorides react with this solvent (Boyle,

1966). The solid and all solutions made from it must be protected from light to avoid photo-

decomposition. Prepare the stock solution immediately before use.

A sulfonyl chloride group rapidly reacts with amines in the pH range of 9–10 to form sta-

ble sulfonamide bonds. Under these conditions, it also may react with tyrosine  OH groups,

aliphatic alcohols, thiols, and histidine side chains. Conjugates of sulfonyl chlorides with sulf-

hydryls and imidazole rings are unstable, while esters formed with alcohols are subject to nucle-

ophilic displacement (Nillson and Mosbach, 1984; Scouten and Van der Tweel, 1984). The only

stable derivative with proteins therefore is the sulfonamide, formed by reaction with -lysine

Figure 9.17 Texas Red sulfonyl chloride can be used to label amine-containing molecules through sulfonamide

bond formation.

and N-terminal amines. For coupling, the reaction media should use only non-amine-containing

buffers, such as phosphate, borate, or bicarbonate (avoid Tris, imidazole, or glycine).

A suggested protocol on the use of this ﬂ uorescent probe is described below. Optimization

may be necessary to achieve the best level of ﬂ uorescent modiﬁ cation ( F / P ratio) for a particu-

lar application.

Protocol

1. Dissolve the protein or macromolecule to be labeled in 0.1 M sodium carbonate, pH 9.0,

at a concentration of 1–5 mg/ml.

2. Dissolve Texas Red sulfonyl chloride (Thermo Fisher, Invitrogen) in acetonitrile at a con-

centration of 20 mg/ml. Prepare fresh and protect from light. Use a fume hood for all

operations using organic solvents.

3. In subdued lighting conditions, add 50 l of the Texas Red sulfonyl chloride solution to

each ml of the protein solution. Mix well.

4. React for 1 hour at room temperature.

5. Remove excess ﬂ uorophore and reaction by-products by gel ﬁ ltration using a desalting

resin or by dialysis.

Determine the level of ﬂ uorophore incorporation (the F / P ratio) by measuring the absorb-

ance of the labeled protein at 520 and 280 nm. Labeled proteins having a 520 nm/280 nm ratio

of absorbency of 0.3–0.8 should perform well in most applications (Titus et al. , 1982).

At the time of this writing, over 15,000 references cited the use of Texas Red for labeling

biological molecules or in bioconjugate detection applications.

Sulfhydryl-Reactive Rhodamine Derivatives

Rhodamine 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).

Tetramethylrhodamine-5-(and 6)-iodoacetamide

The iodoacetamido derivatives of tetramethylrhodamine possess a sulfhydryl-reactive iodoacetyl

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

tion on their lower ring. The isomers are commercially available only in mixed form, but some

reactivity and speciﬁ city differences between the puriﬁ ed 5- and 6-derivatives toward various

sulfhydryl sites in proteins may be observed (Ajtai et al ., 1992) (Invitrogen).

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

ditions to yield stable thioether linkages ( Figure 9.18 ). They do not react with unreduced

disulﬁ des in cystine residues or with oxidized glutathione (Gorman et al., 1987). The thioether

bonds are hydrolyzed under conditions necessary for complete protein hydrolysis prior to

amino acid analysis.

2. Rhodamine Derivatives 425

426 9. Fluorescent Probes

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

yield of the rhodamine derivative, but also to protect the iodoacetyl group from light-catalyzed

breakdown. Tetramethylrhodamine-5-(and-6)-iodoacetamide is soluble in DMF and DMSO.

Concentrated stock solutions may be prepared in these solvents prior to addition of a small aliquot

to an aqueous reaction mixture. Protect solutions from light by wrapping in aluminum foil and

working in subdued light. Quenching reactions with cysteine, glutathione, or mercaptosuccinic

acid will sometimes facilitate removal of unconjugated ﬂ uorophore by dialysis or gel ﬁ ltration.

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

maximum occurs at about 543 nm and its emission peak at 567 nm, producing light in the

orange-red region of the spectrum. The extinction coefﬁ cient of tetramethylrhodamine-5-

(and-6)-iodoacetamide in methanol at its wavelength of maximum absorptivity, 542 nm, is

81,000 M

 1

Figure 9.18 This iodoacetamide derivative of tetramethylrhodamine can be used to label sulfhydryl groups via

thioether bond formation.

The ﬂ uorescent probe has been used extensively to label numerous proteins and other bio-

molecules, including actin (Glacy, 1983; Wang, 1985; Meige and Wang, 1986), myosin light

chains (Mittal et al., 1987), -actin (Simon and Taylor, 1988; Stickel and Wang, 1988), blood

coagulation factor Va (Isaacs et al., 1986), and histones (Murphy et al., 1982). The dye also

has been used to study conformational changes in proteins (Heuck et al., 2007), to study the

binding region of protein C on factor Va (Yegneswaran et al., 2007), and how ﬂ avonoids affect

actin functions (Boehl et al., 2007). Hundreds of additional publications cite the use of this dye

for various biological detection applications.

The following protocol for labeling proteins with tetramethylrhodamine-5-(and-6)-iodoa-

cetamide represents a general guideline. The procedure should be optimized for each macro-

molecule being labeled to obtain the best F / P ratio to produce intense ﬂ uorescence and high

activity in the ﬁ nal complex.

Protocol

1. Prepare a 20 mM tetramethylrhodamine-5-(and-6)-iodoacetamide solution by dissolving

11.3 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 50 mM sodium

phosphate, pH 7.5.

3. Slowly add 25–50 l of the tetramethylrhodamine-5-(and-6)-iodoacetamide 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 by dialysis.

Aldehyde/Ketone and Cytosine-Reactive Rhodamine Derivatives

Hydrazide groups can be coupled directly to aldehydes and ketones to form relatively stable

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

able that contain a sulfonyl hydrazine group off their No. 5 carbon on the lower-ring structure

(Invitrogen). They are based on the Lissamine and Texas Red structures and may be used to

label aldehyde- or ketone-containing molecules with an intensely ﬂ uorescent probe. Although

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

drates, glycoproteins, RNA, and other molecules containing sugar residues (or diols) 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 with hydrazide- or amine-containing probes (Draper and Gold, 1980)

(Chapter 27, Section 2.1).

Lissamine Rhodamine B Sulfonyl Hydrazine

Lissamine rhodamine B sulfonyl hydrazine is a hydrazide derivative of sulforhodamine B that

can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent,

2. Rhodamine Derivatives 427

428 9. Fluorescent Probes

hydrazone linkage ( Figure 9.19 ). 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 556 nm and a maximal emission

wavelength of 580 nm when dissolved in methanol. In the same solvent, the compound has an

extinction coefﬁ cient of approximately 75,000 M

 1

Lissamine rhodamine B sulfonyl hydrazine is soluble in DMF. The reagent may be dissolved

in this solvent 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.

Generalized protocols for the use of hydrazine probes reactive toward aldehyde residues can

be found in Section 1, this chapter. These procedures are directed at the labeling of cell-surface

Figure 9.19 Lissamine rhodamine B sulfonyl hydrazine can react with aldehyde groups to form hydrazone linkages.

glycoproteins or glycoproteins in solution. Substitution of Lissamine rhodamine B sulfonyl

hydrazine for the ﬂ uorescein-5-thiosemicarbazide reagent described in that section can be done

without difﬁ culty. Some optimization may be necessary to achieve the best level of ﬂ uorescent

modiﬁ cation for each particular application.

Texas Red Hydrazide

Texas Red hydrazide is a derivative of Texas Red sulfonyl chloride made by reaction with

hydrazine (Invitrogen). The result is a sulfonyl hydrazine group on the No. 5 carbon position

of the lower-ring structure of sulforhodamine 101. The intense Texas Red ﬂ uorophore has a

QY that is inherently higher than either the tetramethylrhodamine or Lissamine rhodamine B

derivatives of the basic rhodamine molecule. Texas Red ’s luminescence is shifted maximally

into the red region of the spectrum, and its emission peak only minimally overlaps with that of

ﬂ uorescein. This makes derivatives of this ﬂ uorescent probe among the best choices of labels

for use in double-staining techniques.

The hydrazide derivative can be used to modify aldehyde- or ketone-containing molecules,

including cytosine residues using the bisulﬁ te activation procedure described in Chapter 27,

Section 2.1. The sulfonyl hydrazine group of Texas Red hydrazide reacts with aldehydes or

ketones in target molecules to form hydrazone bonds ( Figure 9.20 ). Carbohydrates and glyco-

conjugates can be speciﬁ cally labeled at the polysaccharide portion if the required aldehydes

are ﬁ rst formed by periodate oxidation or another such method (Chapter 1, Section 4.4).

Texas Red hydrazide has a maximal excitation wavelength of 580 nm and a maximum

emission at 604 nm when dissolved in methanol. Its extinction coefﬁ cient in the same solvent

is 80,000 M

 1

at 580 nm. The only disadvantage of this ﬂ uorophore is its poor excita-

tion by an argon laser at 488 nm. However, since both Texas Red and ﬂ uorescein are weakly

excited by an argon laser at 514 nm, it makes them fairly good pairs for use in laser confocal

microscopy or ﬂ ow cytometry (Mossberg and Ericsson, 1990). The ﬂ uorophore is particularly

2. Rhodamine Derivatives 429