Detecting Glycosidases - Section 10.2

Fluorescein Digalactoside

Probably the most sensitive fluorogenic substrate for detecting β-galactosidase is fluorescein di-β-D-galactopyranoside (FDG, F1179; ; Fluorescein Digalactoside). Nonfluorescent FDG is sequentially hydrolyzed by β-galactosidase, first to fluorescein monogalactoside (FMG) and then to highly fluorescent fluorescein (F1300, F36915; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1; ). Enzyme-mediated hydrolysis of FDG can be followed by the increase in either absorbance or fluorescence. Although the turnover rates of FDG and its analogs are considerably slower than that of the common spectrophotometric galactosidase substrate, o-nitrophenyl β-D-galactopyranoside (ONPG), the absorbance of fluorescein is about fivefold greater than that of o-nitrophenol. Moreover, fluorescence-based measurements can be several orders of magnitude more sensitive than absorption-based measurements. Fluorescence-based assays employing FDG are also reported to be 100- to 1000-fold more sensitive than radioisotope-based ELISAs.

In addition to its use in ELISAs, the FDG substrate has proven very effective for identifying lacZ-positive cells with fluorescence microscopy and flow cytometry. FDG has been employed to identify cells infected with recombinant herpesvirus, to detect unique patterns of β-galactosidase expression in live transgenic zebrafish embryos and to monitor β-galactosidase expression in bacteria. The purity of FDG and its analogs is very important because a reagent with extremely low fluorescence background is essential for most applications. Our stringent quality control ensures that the fluorescent contamination of FDG is less than 50 ppm.

The FluoReporter lacZ Flow Cytometry Kits (50-test kit, F1930; 250-test kit, F1931) provide materials and protocols for quantitating β-galactosidase activity with FDG in single cells using flow cytometry. These kits are accompanied by a license to practice patented techniques for loading FDG by hypotonic shock and improving retention of fluorescein in lacZ-positive cells. In addition to a detailed protocol (FluoReporter(R) lacZ Flow Cytometry Kits), each FluoReporter lacZ Flow Cytometry Kit contains convenient premixed solutions of:

• FDG
• Phenylethyl β-D-thiogalactopyranoside (PETG; also available separately as a solid, P1692), a broad-spectrum β-galactosidase inhibitor for stopping the reaction
• Chloroquine diphosphate for inhibiting hydrolysis of the substrate in acidic organelles by endogenous galactosidase enzymes
• Propidium iodide for detecting dead cells

Each kit provides sufficient reagents for 50 (in Kit F1930) or 250 (in Kit F1931) flow cytometry assays. This assay enables researchers to detect heterogeneous expression patterns and to sort and clone individual cells expressing known quantities of β-galactosidase. Practical reviews on using FDG for flow cytometric analysis and sorting of lacZ-positive cells are available.

The fluorescent hydrolysis product of FDG is fluorescein (F1300, F36915; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1; Figure 15.3), which rapidly leaks from cells under physiological conditions, making the use of FDG problematic for prolonged studies. Our DetectaGene Green, ImaGene Green and ImaGene Red substrates (see below) have been specifically designed to improve retention of the fluorescent hydrolysis products in cells.

Figure 15.3 Loading and retention characteristics of intracellular marker dyes. Cells of a human lymphoid line (GePa) were loaded with the following cell-permeant acetoxymethyl ester (AM) or acetate derivatives of fluorescein: 1) calcein AM (C1430, C3099, C3100MP), 2) BCECF AM (B1150), 3) fluorescein diacetate (FDA, F1303), 4) carboxyfluorescein diacetate (CFDA, C1354) and 5) CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, C2925, C7025). Cells were incubated in 4 µM staining solutions in Dulbecco's modified eagle medium containing 10% fetal bovine serum (DMEM+) at 37°C. After incubation for 30 minutes, cell samples were immediately analyzed by flow cytometry to determine the average fluorescence per cell at time zero (0 hours). Retained cell samples were subsequently washed twice by centrifugation, resuspended in DMEM+, maintained at 37°C for 2 hours and then analyzed by flow cytometry. The decrease in the average fluorescence intensity per cell in these samples relative to the time zero samples indicates the extent of intracellular dye leakage during the 2-hour incubation period.

Resorufin Galactoside

Unlike FDG, resorufin β-D-galactopyranoside (R1159) requires only a single-step hydrolysis reaction to attain full fluorescence. This substrate is especially useful for sensitive enzyme measurements in ELISAs. The relatively low pK_a (~6.0) of its hydrolysis product, resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1, ), permits its use for continuous measurement of enzymatic activity at physiological pH. Resorufin galactoside has also been used to quantitate β-galactosidase activity in single yeast cells by flow cytometry and to detect immobilized β-galactosidase activity in bioreactors.

DDAO Galactoside

Although substrates based on DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)) are intrinsically fluorescent (excitation/emission ~460/610 nm), β-galactosidase–catalyzed hydrolysis of DDAO galactoside (D6488) liberates the DDAO fluorophore (), which absorbs and emits light at much longer wavelengths (excitation/emission ~645/660 nm) (Figure 10.13). Not only can DDAO (H6482, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1) be excited without interference from the unhydrolyzed substrate, but its fluorescence emission is detected at wavelengths that are well beyond the autofluorescence exhibited by most biological samples. The relatively low pK_a of DDAO (~5.5) permits continuous monitoring of β-galactosidase activity at physiological pH.

Figure 10.13 Absorption spectra of 1) DDAO galactoside (D6488) and 2) DDAO (H6482) at equal concentrations in pH 9 aqueous buffer. These spectra show the large spectral shift accompanying enzymatic cleavage of DDAO-based substrates. DDAO phosphate (D6487) has very similar spectra.

Methylumbelliferyl Galactoside and Its Difluorinated Analog

The fluorogenic β-galactosidase substrate β-methylumbelliferyl β-D-galactopyranoside (MUG, M1489MP) is commonly used to detect β-galactosidase activity in cell extracts, lysosomes and human blood serum. However, the hydrolysis product, 7-hydroxy-4-methylcoumarin (β-methylumbelliferone, H189; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1; Figure 10.2), has a relatively high pK_a (~7.8), precluding its use for continuous measurement of enzymatic activity. 6,8-Difluoro-4-methylumbelliferyl β-D-galactopyranoside (DiFMUG, D11945) yields a hydrolysis product — 6,8-difluoro-7-hydroxy-4-methylcoumarin (D6566, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1, Figure 10.2) — with a much lower pK_a (4.9), allowing its detection at physiological pH. Given the low pK_a of its hydrolysis product, DiFMUG should be especially useful for the continuous in vitro assay of β-D-galactosidase activity at any pH >6. The fluorinated coumarin glycosides are Patented by Molecular Probes.

Figure 10.2 Absorption and fluorescence emission spectra of 7-hydroxy-4-methylcoumarin (H189) in pH 9.0 buffer. The spectra of 6,8-difluoro-7-hydroxy-4-methylcoumarin (D6566) are essentially identical.

Carboxyumbelliferyl Galactoside and the FluoReporter lacZ/Galactosidase Quantitation Kit

Hydrolysis of 3-carboxyumbelliferyl β-D-galactopyranoside (CUG, C1488) by β-galactosidase yields 7-hydroxycoumarin-3-carboxylic acid (H185, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1). 7-Hydroxycoumarin has a pK_a below the pH at which the turnover rate is optimal, facilitating the use of CUG for continuous measurements of β-galactosidase activity. Unlike most substrates for β-galactosidase, CUG is quite water-soluble and can be used over a wide range of concentrations in enzymatic activity measurements. Our FluoReporter lacZ/Galactosidase Quantitation Kit (F2905) provides a CUG-based method for quantitating β-galactosidase activity in ELISAs or lacZ-positive cell extracts. Each kit contains:

• CUG
• 7-Hydroxycoumarin-3-carboxylic acid, a reference standard
• Detailed protocols (FluoReporter(R) lacZ/Galactosidase Quantitation Kit) suitable for use with any fluorescence microplate reader

Sufficient reagents are provided for approximately 1000 β-galactosidase assays. We have demonstrated a practical detection limit of ~0.5 pg of β-galactosidase using this kit and a fluorescence microplate reader.

Fluorescent Glycosphingolipids

β-Galactosidase enzymes that act on the lipophilic sphingosyl galactosides, including galactosylceramidase (EC 3.2.1.46) and G_M1 ganglioside β-galactosidase (EC 3.2.1.23), are particularly important in neurochemistry. The preferred substrates for these enzymes are sphingolipids derived from galactose (Sphingolipids, Steroids, Lipopolysaccharides and Related Probes - Section 13.3). Galactosylceramidase converts substrates such as our BODIPY FL C₁₂-galactosylceramide (D7519) back to the ceramide. Purified G_M1 ganglioside galactosidase removes the terminal galactose residue from lactosylceramides such as our BODIPY FL C₅-lactosylceramide (D13951), yielding the corresponding glucosylceramide. BODIPY FL C₅-lactosylceramide complexed with bovine serum albumin (BSA) (B34402) may also be useful for assaying G_M1 ganglioside galactosidase. Complexing fluorescent lipids with BSA facilitates the preparation of aqueous solutions by eliminating the need for organic solvents to dissolve the lipophilic probe. The lack of a spectral shift between the substrates and the hydrolysis products in these reactions means that extraction and chromatographic separation of the products is necessary for assessment of enzyme activity.

Our Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, A12222, A22177; Substrates for Oxidases, Including Amplex Red Kits - Section 10.5; ) is an unusually stable peroxidase substrate that we have used in coupled reactions to detect a wide variety of analytes, including both enzymes and their substrates (see Substrates for Oxidases, Including Amplex Red Kits - Section 10.5 for a list of all of our Amplex Red Kits and reagents). Most of the assays can be performed as continuous assays at neutral or slightly acidic pH and are particularly suitable for automation and high-throughput screening using either an absorption- or fluorescence-based microplate reader.

Rather than requiring an unnatural fluorogenic or chromogenic substrate for β-galactosidase (or α-galactosidase), our Amplex Red reagent–based technology permits the direct quantitation of free galactose, which is produced by a wide variety of enzymes. Even enzymes that act on polysaccharides and glycolipids but are difficult to assay using known chromogenic substrates can, in some cases, be detected and their activity quantitated using the Amplex Red reagent in combination with galactose oxidase and horseradish peroxidase. Unlike glucose oxidase, galactose oxidase produces H₂O₂ from either free galactose or from polysaccharides — including glycoproteins in solution and on cell surfaces — and from certain glycolipids in which galactose is the terminal residue (Figure 10.14). Because the galactose oxidase–catalyzed reaction does not require prior cleavage of the glycoside to free galactose by a galactosidase, appropriate control reactions must be used to ascertain whether the rate-limiting step is the galactosidase- or galactose oxidase–mediated reaction.

The Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179) provides an ultrasensitive method for detecting galactose (Figure 10.15) and galactose oxidase (Figure 10.16) activity. This assay utilizes the Amplex Red reagent () to detect H₂O₂ generated by galactose oxidase–mediated oxidation of desialated galactose moieties. In the presence of horseradish peroxidase (HRP), the H₂O₂ thus produced reacts with the Amplex Red reagent in a 1:1 stoichiometry to generate the red-fluorescent oxidation product resorufin. Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively (), and because its extinction coefficient is high (54,000 cm^-1M^-1), the assay can be performed either fluorometrically or spectrophotometrically. The Amplex Red Galactose/Galactose Oxidase Assay Kit provides all the reagents and a general protocol for the assay of galactose-producing enzymes or for the assay of galactose oxidase, including:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• Galactose oxidase from Dactylium dendroides
• D-Galactose
• Detailed protocols (Amplex(R) Red Galactose/Galactose Oxidase Kit)

Sufficient reagents are provided for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and reaction volumes of 100 µL per assay. The Amplex Red galactose/galactose oxidase assay accurately measures as low as 4 µM galactose and 2 mU/mL galactose oxidase activity (Figure 10.15, Figure 10.16). Because of the high absorbance of resorufin, the absorptimetric assay has only slightly lower sensitivity than the fluorometric assay.

Figure 10.14 Detection scheme utilized in the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Oxidation of the terminal galactose residue of a glycoprotein, glycolipid or polysaccharide results in the generation of H₂O₂, which, in the presence of horseradish peroxidase (HRP), reacts stoichiometrically with the Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin.



Figure 10.15 Detection of galactose using the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Each reaction contained 50 µM Amplex Red reagent, 0.1 U/mL HRP, 2 U/mL of galactose oxidase and the indicated amount of galactose in 1X reaction buffer. Reactions were incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. A background fluorescence of 93 units was subtracted from each data point.



Figure 10.16 Detection of galactose oxidase activity using the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Each reaction contained 50 µM Amplex Red reagent, 0.1 U/mL HRP, 100 µM galactose and the indicated amount of galactose oxidase in 1X reaction buffer. Reactions were incubated at 37°C. After 20 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm with fluorescence detection at 590 ± 17.5 nm.

The primary problems associated with detecting lacZ expression in live cells using fluorogenic substrates are:

• Difficulty in loading the substrates under physiological conditions
• Leakage of the fluorescent product from live cells
• High levels of endogenous β-galactosidase activity in many cells

Our DetectaGene Green, ImaGene Green and ImaGene Red Kits are designed to improve the sensitivity of β-galactosidase assays by yielding products that are better retained in viable cells and, in the case of the ImaGene Green and ImaGene Red Kits, by providing substrates that can be passively loaded into live cells. The high level of endogenous β-galactosidase activity remains an obstacle when detecting low levels of lacZ expression.

DetectaGene Green lacZ Gene Expression Kit

The Patented substrate in our DetectaGene Green lacZ Gene Expression Kit (D2920) — 5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG) — is a galactose derivative that has been chemically modified to include a mildly thiol-reactive chloromethyl group (Figure 10.17). Once loaded into the cell using the Influx pinocytic cell-loading reagent (I14402, included in Kit D2920; ) or by microinjection, hypotonic shock or another technique (Techniques for loading molecules into the cytoplasm - Table 14.1), the DetectaGene substrate undergoes two reactions: 1) its two galactose moieties are cleaved by intracellular β-galactosidase and 2) either simultaneously or sequentially, its chloromethyl moiety reacts with glutathione and possibly other intracellular thiols to form a membrane-impermeant, peptide–fluorescent dye adduct (Figure 10.17). Because peptides do not readily cross the plasma membrane, the resulting fluorescent adduct is much better retained than is the free dye, even in cells that have been kept at 37°C. We have found that lacZ-positive cells loaded from medium containing 1 mM CMFDG are as fluorescent as cells loaded with 40-fold higher concentrations of FDG. Furthermore, unlike the free dye, the peptide–fluorescent dye adducts contain amine groups and can therefore be covalently linked to surrounding biomolecules by fixation with formaldehyde or glutaraldehyde. This property permits long-term storage of the labeled cells or tissue, as well as amplification of the conjugate by standard immunohistochemical techniques using our anti-fluorescein/Oregon Green antibody (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Anti-fluorophore antibodies and their conjugates - Table 7.19).

The CMFDG substrate in our DetectaGene Green lacZ Gene Expression Kit was used to stain lacZ-expressing floor plate cells in tissue dissected from a developing mouse embryo, to identify lacZ-enhancer–trapped Drosophila neurons in culture and to detect β-galactosidase activity in hippocampus slices. In the latter study, the fluorescence of the neurons could still be visualized 24 hours after dye loading, and the fluorescent CMFDG-loaded neurons exhibited a normal pattern and time course of axonal outgrowth and branching. CMFDG also has been microinjected into primary hepatocytes, fibroblasts and glioma cells to detect β-galactosidase activity and has been incorporated into an electrophysiological recording pipette to confirm the identity of neurons cotransfected with the lacZ gene and a second gene encoding Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II).

The DetectaGene Green CMFDG lacZ Gene Expression Kit (D2920) contains:

• DetectaGene Green CMFDG substrate (Figure 10.17)
• Phenylethyl β-D-thiogalactopyranoside (PETG; also available separately as a solid, P1692), a broad-spectrum β-galactosidase inhibitor for stopping the reaction
• Verapamil for inhibiting product efflux
• Chloroquine diphosphate for inhibiting acidic hydrolysis of the substrate
• Propidium iodide for detecting dead cells
• Influx pinocytic cell-loading reagent for introducing CMFDG into cells
• Detailed protocols for detecting β-galactosidase activity (DetectaGene Green CMFDG lacZ Gene Expression Kit)

When used at the recommended dilutions, sufficient reagents are provided for approximately 200 flow cytometry tests with the DetectaGene Green CMFDG Kit. Verapamil has been added to the DetectaGene Green CMFDG lacZ Gene Expression Kit because we have observed that cell retention of the fluorescent dye–peptide adduct can be considerably improved in many cell types by adding verapamil to the medium.

Figure 10.17 Sequential β-galactosidase hydrolysis and peptide conjugate formation of CMFDG, a component of the DetectaGene Green CMFDG lacZ Gene Expression Kit (D2920).

PFB Aminofluorescein Digalactoside

Our Patented 5-(pentafluorobenzoylamino)fluorescein di-β-D-galactopyranoside (PFB-FDG, P11948; ) yields the green-fluorescent PFB-F dye (P12925, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1), which appears to localize to endosomal and lysosomal compartments when loaded into cells by pinocytosis (), similar to our PFB aminofluorescein diglucoside (PFB-FDGlu, P11947; see below). Thus, PFB-FDG is potentially useful for studying lysosomal storage diseases, including Krabbe's disease, G_M1 gangliosidosis, galactosialidosis and Morquio syndrome, which are all associated with deficient lysosomal β-galactosidase activity.

ImaGene Green and ImaGene Red lacZ Reagents and Gene Expression Kits

The Patented fluorescein- and resorufin-based galactosidase substrates in our ImaGene Green and ImaGene Red lacZ Gene Expression Kits (I2904, I2906) have been covalently modified to include a 12-carbon lipophilic moiety. Unlike FDG or CMFDG (Figure 10.17), these lipophilic fluorescein- and resorufin-based substrates — abbreviated C₁₂FDG () and C₁₂RG () for the ImaGene Green and ImaGene Red substrates, respectively — can be loaded simply by adding the substrate to the aqueous medium in which the cells or organisms are growing, either at ambient temperatures or at 37°C. Once inside the cell, the substrates are cleaved by β-galactosidase, producing fluorescent products that are well retained by the cells, probably by incorporation of their lipophilic tails within the cellular membranes. Mammalian NIH 3T3 lacZ-positive cells grown for several days in medium containing 60 µM C₁₂FDG appear morphologically normal, continue to undergo cell division and remain fluorescent for up to three cell divisions after replacement with substrate-free medium.

The C₁₂FDG substrate in our ImaGene Green lacZ Expression Kit (I2904) is superior to FDG for flow cytometric detection of β-galactosidase activity in live mammalian cells. Using C₁₂FDG with flow cytometric methods, researchers have:

• Assessed levels of lacZ gene expression in recombinant Chinese hamster ovary (CHO) cells throughout the cell cycle, which was monitored with Hoechst 33342 (H1399; H3570; FluoroPure Grade - Note 19.2, H21492; Nucleic Acid Stains - Section 8.1)
• Identified endocrine cell precursors in dissociated fetal pancreatic tissue based on their high levels of endogenous acid β-galactosidase
• Measured β-galactosidase activity in single recombinant E. coli bacteria
• Detected the activity of β-galactosidase fusion proteins in yeast
• Sorted β-galactosidase–expressing mouse sperm cells and insect cells that harbor recombinant baculovirus

The C₁₂FDG substrate was also useful in a fluorescence microscopy study of zebrafish expressing a lacZ reporter gene that was under the control of a mammalian homeobox gene promoter. In some cell types, C₁₂FDG produces high levels of background fluorescence that may prohibit its use in assaying low β-galactosidase expression.

Molecular Probes' ImaGene Green C₁₂FDG or ImaGene Red C₁₂RG lacZ Gene Expression Kits contain:

• ImaGene Green C₁₂FDG (in Kit I2904) or ImaGene Red C₁₂RG (in Kit I2906)
• Phenylethyl β-D-thiogalactopyranoside (PETG; also available separately as a solid, P1692), a broad-spectrum β-galactosidase inhibitor for stopping the reaction
• Chloroquine diphosphate for inhibiting acidic hydrolysis of the substrate
• Detailed protocols for detecting β-galactosidase activity (ImaGene Green C{12}FDG lacZ Gene Expression Kit, ImaGene Red C{12}RG lacZ Gene Expression Kit)

Each kit provides sufficient reagents for 100–200 assays, depending on the volume used for each experiment.

5-Dodecanoylaminofluorescein di-β-D-galactopyranoside (C₁₂FDG) is available separately (D2893) and we also offer the somewhat less lipophilic 5-octanoylaminofluorescein di-β-D-galactopyranoside (C₈FDG, O2892). The C₈FDG analog is optimal for investigating the expression of lacZ fusion genes in sporulating cultures of Bacillus subtilis and is a better substrate than C₁₂FDG for the detection of β-galactosidase activity in sperm containing the lacZ gene. 5-Acetylaminofluorescein di-β-D-galactopyranoside (C₂FDG, A22010) is particularly useful for detecting lacZ reporter gene expression in slow-growing mycobacteria, including Mycobacterium tuberculosis, using a fluorescence microplate reader or a flow cytometer.

Chloromethyl and Lipophilic Derivatives of DiFMUG

The relatively low pK_a of our 6,8-difluoro-7-hydroxycoumarin derivatives (Figure 1.95) has also allowed us to develop some useful probes for detecting enzymatic activity in vivo. Although the β-galactosidase DiFMUG (D11945) readily enters many live eukaryotic cells, its hydrolysis product (6,8-difluoro-7-hydroxy-4-methylcoumarin, DiFMU; D6566; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1; ) is not well retained. To address this limitation, we have developed two modified galactosidase substrates using product-retention strategies that have proven useful for our Patented DetectaGene Green, ImaGene Green and ImaGene Red glycosidase substrates.

Figure 1.95 Comparison of the pH-dependent fluorescence changes produced by attachment of electron-withdrawing fluorine atoms to a hydroxycoumarin. 7-Hydroxy-4-methylcoumarin-3-acetic acid (, H1428) and 6,8-difluoro-7-hydroxy-4-methylcoumarin (, D6566) demonstrate the decrease of the pK_a from ~7.4 to ~6.2. Fluorescence intensities were measured for equal concentrations of the two dyes using excitation/emission at 360/450 nm.

As with our DetectaGene products, we have replaced the methyl group of DiFMUG with a mildly thiol-reactive chloromethyl group, yielding the β-galactosidase substrate CMDiFUG (C11946), which is the 6,8-difluorinated analog of 4-chloromethylcoumarin-7-yl β-D-galactopyranoside (CMCG). We have previously shown that incorporating a chloromethyl group into dyes, as in our CellTracker and MitoTracker probes, considerably improves the retention of fluorescent products in live cells. This enhanced cell retention is at least partially attributable to the formation of dye conjugates with intracellular thiols, including glutathione. Our results indicate that CMDiFUG discriminates lacZ-positive and lacZ-negative live cells better than all the other fluorogenic β-galactosidase substrates we have tested, including our DetectaGene Green, ImaGene Green and ImaGene Red substrates.

Similar to our ImaGene Green and ImaGene Red products, 6,8-difluoro-4-heptadecylumbelliferyl β-D-galactopyranoside (C₁₇DiFUG, D11950) contains a lipophilic moiety in place of the methyl group of DiFMUG (). This modification improves the penetration of this substrate through cell membranes, as well as the retention of the fluorescent product of β-galactosidase activity in live cells. The fluorinated coumarin glycosides are Patented by Molecular Probes.

The substrate 4-methylumbelliferyl β-D-glucuronide (MUGlcU, M1490) is probably the most commonly used fluorogenic reagent for identifying E. coli contamination and for detecting GUS reporter gene expression in plants and plant extracts. However, β-glucuronidase substrates based on fluorescein may be much more sensitive and yield products that are fluorescent at physiological pH, making them useful for continuous monitoring of enzymatic activity. In addition, we offer a fluorogenic ELF 97 β-D-glucuronidase substrate (E6587), which produces an intensely green-fluorescent precipitate at the site of enzymatic activity that can be clearly distinguished from most autofluorescence. This substrate has been used for in-gel zymography to detect β-glucuronidase activity (Figure 9.29, Figure 10.24), immunoassays on protein microarrays and for the flow cytometric analysis and separation of E. coli that had been transfected with gusA expression vectors.

Figure 9.29 SYPRO Tangerine protein gel stain and ELF β-D-glucuronide for zymography. Molecular weight standards containing decreasing amounts of Escherichia coli β-glucuronidase were run on an SDS-polyacrylamide gel and stained with SYPRO Tangerine protein gel stain (orange, S12010) followed by incubation with the ELF 97 β-D-glucuronidase substrate (green, E6587) to detect the activity of β-glucuronidase.

Figure 10.24 In situ gel assay of β-D-glucuronidase (GUS) activity with ELF 97 β-D-glucuronide (E6587). A) Twofold dilutions of the purified GUS enzyme or B) cell extracts from single leaves from GUS-positive and -negative Arabidopsis plants were electrophoresed through a native 7.5% polyacrylamide gel. Following electrophoresis, the gel was washed with 0.1 M sodium phosphate, pH 7.0, containing 0.2% Triton X-100, at room temperature for 60 minutes and then incubated with 15 µM ELF 97 β-D-glucuronide in 0.1 M sodium phosphate, pH 7.0, at 37°C, for 30–60 minutes. The gel was photographed using 300 nm transillumination, a SYBR photographic filter (S7569, Accessories for Electrophoresis - Section 23.4) and Polaroid 667 black-and-white print film.

Fluorescein Diglucuronide

Fluorescein di-β-D-glucuronide (FDGlcU, F2915) is colorless and nonfluorescent until it is hydrolyzed to the monoglucuronide and then to highly fluorescent fluorescein (F1300, F36915; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1). FDGlcU has been used to detect β-glucuronidase activity in plant extracts containing the GUS reporter gene and may also be useful for assaying lysosomal enzyme release from neutrophils. FDGlcU has also been used in the flow cytometric assay of individual mammalian cells expressing the E. coli β-glucuronidase gene.

PFB Aminofluorescein Diglucuronide

Our Patented 5-(pentafluorobenzoylamino)fluorescein di-β-D-glucuronide (PFB-FDGlcU, P11949) yields the green-fluorescent PFB-F (P12925, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1), which appears to localize to endosomal and lysosomal compartments when loaded into cells by pinocytosis, similar to our PFB aminofluorescein diglucoside (PFB-FDGlu, P11947). PFB-FDGlcU has been used for the quantitative analysis of β-glucuronidase activity in viable cells and for sorting high-expressing cells by flow cytometry. Enzyme enrichment has promise as a tool for gene therapy.

Coumarin Glucuronides

4-Methylumbelliferyl β-D-glucuronide (MUGlcU, M1490) has been used extensively to detect E. coli in food, water, urine and environmental samples. MUGlcU is stable to the conditions required for sterilization of media. A fluorogenic bioassay using MUGlcU has been developed to assess the detrimental effects of Li⁺, Al³⁺, Cr⁶⁺ and Hg²⁺ on the proliferation of E. coli.

MUGlcU is also commonly used to identify plant tissue expressing the GUS reporter gene, including nondestructive assays that allow propagation of the transformed plant lines. In addition, MUGlcU has served as a sensitive substrate for lysosomal enzyme release from neutrophils.

Enzyme-mediated hydrolysis of 6,8-difluoro-4-methylumbelliferyl β-D-glucuronide (DiFMUGlcU, D11951) yields a highly fluorescent product (6,8-difluoro-7-hydroxy-4-methylcoumarin, DiFMU; D6566; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1; ) that has a very low pK_a, which should make DiFMUGlcU especially useful for the continuous in vitro assay of β-D-glucuronidase activity at pH 6 or greater. The hydrolysis product of β-trifluoromethylumbelliferyl β-D-glucuronide (T658) exhibits longer-wavelength excitation and emission spectra than those of either MUGlcU or DiFMUGlcU, which can be advantageous for cells that have high levels of endogenous fluorescence, such as plant cells.

ImaGene Green β-D-Glucuronidase Substrate

Molecular Probes also offers a lipophilic analog of fluorescein di-β-D-glucuronide in our ImaGene Green C₁₂FDGlcU GUS Gene Expression Kit (I2908). As with our similar ImaGene Green and ImaGene Red substrates for β-galactosidase (see above), we have shown that this lipophilic β-glucuronidase substrate freely diffuses across the membranes of viable cultured tobacco leaf cells or protoplasts under physiological conditions. Furthermore, the fluorescent cleavage product is retained in the plant cell for hours to days, facilitating long-term measurements of GUS gene expression. In thin sections of transgenic tomato leaf, the ImaGene Green C₁₂FDGlcU GUS Gene Expression Kit provided a simple and reliable GUS assay that, coupled with confocal laser-scanning microscopy, yielded good cellular resolution. The substrate has also been used to detect β-glucuronidase activity in an Acremonium transformant containing the GUS reporter gene.

Molecular Probes' ImaGene Green C₁₂FDGlcU GUS Gene Expression Kit contains:

• ImaGene Green C₁₂FDGlcU
• D-Glucaric acid-1,4-lactone, a β-glucuronidase inhibitor for stopping the reaction
• Detailed protocols for detecting β-glucuronidase activity (ImaGene Green C{12}FDGlcU GUS Gene Expression Kit)

Each kit provides sufficient reagents for approximately 100 tests, depending on the volume used for each experiment.

ELF 97 β-D-Glucuronide

Molecular Probes' ELF 97 β-D-glucuronidase substrate (ELF 97 β-D-glucuronide, E6587) may be the ideal substrate for analyzing GUS enzyme activity in transgenic plants and in fixed cells and tissues. Upon hydrolysis, this fluorogenic substrate produces a bright yellow-green–fluorescent precipitate at the site of enzymatic activity. This fluorescent precipitate has some unique spectral characteristics, including an extremely large Stokes shift (Figure 6.17), that make it easily distinguishable from the endogenous fluorescent components commonly found in plants (see Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3 for a description of our Patented ELF technology). We have used this substrate to detect the GUS enzyme in Arabidopsis and have found that, after only 4 hours incubation, the signal can be detected in whole-leaf cuttings from GUS-positive plants. Homogenization of a small portion of a leaf from a GUS-positive Arabidopsis plant followed by separation on a nondenaturing gel yields a discrete band corresponding to the glucuronidase enzyme (Figure 10.24). We have also used the ELF 97 glucuronidase substrate for in-gel zymography in one aspect of our Multiplexed Proteomics technology (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4, Figure 9.29). It is possible to detect as little as 0.5 ng of purified β-glucuronidase in a nondenaturing gel incubated with ELF 97 glucuronide. This substrate may also be useful for detecting GUS fusion proteins in gels, for identifying E. coli in agarose-containing medium and for assaying lysosomal enzyme release from neutrophils. The ELF substrates are Patented by Molecular Probes.

Figure 6.17 The normalized fluorescence excitation and emission spectra of the ELF 97 alcohol precipitate (E6578), which is generated by enzymatic cleavage of the soluble ELF 97 phosphatase substrate (E6588, E6589).

β-Glucosidase, which is a marker for the endoplasmic reticulum (Probes for the Endoplasmic Reticulum and Golgi Apparatus - Section 12.4), is present in nearly all species. Its natural substrate is a glucosylceramide (Sphingolipids, Steroids, Lipopolysaccharides and Related Probes - Section 13.3). People with Gaucher's disease have mutations in the acid β-glucosidase gene that result in abnormal lysosomal storage. Enzyme replacement therapy in Gaucher's disease patients requires sensitive and selective methods for measuring β-glucosidase activity (Glycosidase enzymes and their fluorogenic and chromogenic substrates - Table 10.1). Plant β-glucosidases are implicated in a variety of key metabolic events and growth-related responses.

Fluorescein Diglucoside

As with the other fluorescein diglycosides, Molecular Probes' fluorogenic fluorescein di-β-D-glucopyranoside (FDGlu, F2881) is likely to yield the greatest sensitivity for detecting β-glucosidase activity in both cells and cell extracts. This substrate has been used to demonstrate the utility of Saccharomyces cerevisiae and Candida albicans exo-1,3-β-glucanase genes as reporter genes. Because these reporter genes encode secreted proteins, assays for reporter gene expression do not require cell permeabilization. FDGlu has been reported to be a selective substrate for the flow cytometric assay of lysosomal glucocerebrosidase activity in a variety of cells. The assay demonstrated the inordinately low glucocerebrosidase activity present in fibroblasts of Gaucher's disease patients.

PFB Aminofluorescein Diglucoside

Through a collaboration with Matthew Lorincz and Leonard A. Herzenberg at Stanford University Medical School, our Patented PFB aminofluorescein diglucoside (PFB-FDGlu, P11947) has proven to be an excellent substrate for the flow cytometric discrimination of normal peripheral blood mononuclear cells (PBMC) from the PBMC of patients with Gaucher's disease, a genetic deficiency in lysosomal β-glucocerebrosidase activity. These researchers loaded the nonfluorescent PFB-FDGlu substrates into cells by pinocytosis, and then observed the green-fluorescent hydrolysis products in endosomal and lysosomal compartments. Under similar loading conditions, we have shown that the hydrolysis products of PFB aminofluorescein digalactoside (PFB-FDG, P11948; ) and of PFB aminofluorescein diglucuronide (PFB-FDGlcU, P11949) are similarly localized to endosomes and lysosomes.

Fluorescent Glucocerebrosides

The natural substrates for glucocerebrosidase are sphingosyl β-D-glucopyranosides. Our BODIPY FL analogs of this molecule — BODIPY FL C₅-glucocerebroside (D7548) and BODIPY FL C₁₂-glucocerebroside (D7547) — are likely to be substrates for this lysosomal enzyme, which is lacking in Gaucher's disease patients. However, the lack of spectral shift of the hydrolysis products — BODIPY FL C₅-ceramide (D3521, Fatty Acid Analogs and Phospholipids - Section 13.2) and BODIPY FL C₁₂-ceramide — means that extraction and chromatographic separation of the products is necessary for assessment of the activity.

Amplex Red Reagent for Glucose

The Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, A12222, A22177; Substrates for Oxidases, Including Amplex Red Kits - Section 10.5; ) is a colorless, stable and extremely versatile peroxidase substrate. In an application similar to our use of the Amplex Red reagent to detect galactose-producing enzymes (see above), we have shown that it is practical to detect free glucose with high specificity at levels as low as 0.5 µg/mL using the Amplex Red reagent in combination with excess glucose oxidase (Figure 10.25). Because the peroxidase- and glucose oxidase–mediated reactions can be coupled, it is potentially possible to measure the release of glucose by any glucosidase enzyme — for instance, α-glucosidase, β-glucosidase and glucocerebrosidase — in either a continuous or discontinuous assay (Figure 10.59). This assay should also be very useful for quantitation of glucose levels in foods, fermentation media and bodily fluids. The long-wavelength spectral properties of resorufin () and high sensitivity of the assay result in little interference from colored components in the samples.

Figure 10.25 Detection of glucose using the Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189). Reactions containing 50 µM Amplex Red reagent, 0.1 U/mL HRP, 1 U/mL glucose oxidase and the indicated amount of glucose in 50 mM sodium phosphate buffer, pH 7.4, were incubated for one hour at room temperature. Fluorescence was then measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Background fluorescence (arbitrary units), determined for a no-glucose control reaction, has been subtracted from each value. The inset shows the sensitivity and linearity of the assay at low levels of glucose (0–15 µM).

Amplex Red Glucose/Glucose Oxidase Assay Kit

Our Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189) provides all the reagents required for the assay of glucose and enzymes that produce glucose. This kit is also useful for the assay of glucose oxidase activity from cell extracts. We have even shown that the Amplex Red reagent can detect glucose liberated from native dextrans by dextranase and from carboxymethylcellulose. The Amplex Red Glucose/Glucose Oxidase Assay Kit contains:

• Amplex Red reagent
• DMSO
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂) for use as a positive control
• Concentrated reaction buffer
• Glucose oxidase
• D-glucose
• Detailed protocols (Amplex(R) Red Glucose/Glucose Oxidase Assay Kit)

The kit provides sufficient reagents for approximately 500 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

Neuraminidase (NA, also known as sialidase) is a very common enzyme that hydrolyzes terminal sialic acid residues on polysaccharide chains, most often exposing a galactose residue. Although NA is found in mammals, it is predominantly expressed in microorganisms such as bacteria and viruses, including the negative-stranded RNA influenza virus. NA located on the surface of the influenza virus is thought to play a key role in its invasion of target cells and subsequent replication through cleavage of target cell receptor sialic acid moieties. Anti-influenza drug design has therefore focused on the inhibition of NA. Various methods using chemiluminescence, absorption and fluorescence have been developed to quantitate NA in biological fluids for detecting influenza virus and for screening inhibitors of NA activity in drug development. The ultimate goal has been to develop a rapid, single-step assay that is sensitive and adaptable for a high-throughput screening format.

Designed to meet these needs, the Amplex Red Neuraminidase (Sialidase) Assay Kit (A22178) provides an ultrasensitive method for detecting NA activity. This assay utilizes the Amplex Red reagent to detect H₂O₂ generated by galactose oxidase–mediated oxidation of desialated galactose, the end result of NA action. In the presence of HRP, the H₂O₂ thus produced reacts with a 1:1 stoichiometry with the Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin. Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively (), and because the extinction coefficient is high (54,000 cm^-1M^-1), the assay can be performed either fluorometrically or spectrophotometrically. In a purified system with fetuin as the substrate, NA levels as low as 0.2 mU/mL have been detected with the Amplex Red Neuraminidase (Sialidase) Assay Kit (Figure 10.26). NA activity can also be detected in biological samples such as serum (Figure 10.27). Kit contents include:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• Galactose oxidase from Dactylium dendroides
• Fetuin from fetal calf serum
• Neuraminidase from Clostridium perfringens
• Detailed protocols (Amplex(R) Red Neuraminidase (Sialidase) Assay Kit)

Each kit provides sufficient reagents for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and reaction volumes of 100 µL per assay.

Chitin is the second most abundant organic compound in nature and various chitinases and N-acetylglucosaminidases are widely distributed in bacteria, plants and eukaryotic cells. We have utilized our proprietary ELF technology (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3) to prepare the ELF 97 chitinase/N-acetylglucosaminidase substrate (ELF 97 N-acetylglucosaminide, ELF 97 NAG; E22011), which is designed to allow spatially resolved detection of enzyme activity on colony indicator plates and histochemical analysis specimens. Other fluorogenic substrates for these enzymes generate diffusible products and are therefore unsuitable for applications of this type. In addition to the capacity for localized precipitation at sites of enzymatic activity, the ELF 97 alcohol product that is generated upon hydrolysis of ELF 97 NAG is extremely photostable (Figure 6.18) and has widely separated fluorescence excitation and emission peaks (~360/520 nm, Figure 6.17). These properties make the signal easy to discriminate from any background fluorescence. ELF 97 NAG has been utilized to differentiate chitinase-active and non-chitinase–active subpopulations of a marine bacterium during chitin degradation.

α-Amylase is a hydrolytic enzyme that catalyzes the conversion of starch to a mixture of glucose, maltose, maltotriose and dextrins. The levels of α-amylase in various fluids of the human body are of clinical importance in the diagnosis of disease states, including pancreatitis and diabetes. Plant and microbial α-amylases are important enzymes for industrial applications ranging from the manufacture of baked goods and dairy products to the production of ethanol and paper.

The EnzChek Ultra Amylase Assay Kit (E33651) provides a solution-based assay with the speed, high sensitivity and convenience required for measuring amylase activity or for screening amylase inhibitors in a high-throughput format. This EnzChek Kit contains a starch derivative — the DQ starch substrate — that is labeled with BODIPY FL dye to such a degree that the fluorescence is quenched (Figure 10.56). Amylase-catalyzed hydrolysis of this substrate relieves the quenching, yielding brightly fluorescent BODIPY FL dye–labeled fragments. The accompanying increase in fluorescence is proportional to amylase activity and can be monitored with a fluorescence microplate reader or fluorometer, using standard fluorescein filters. In tests using α-amylase from Bacillus sp. (Sigma A-6380) and 200 g/mL of the DQ starch substrate (30-minute incubation at room temperature), the EnzChek Ultra Amylase Assay Kit could be used to detect α-amylase activity down to a final concentration of 2 × 10^-3 U/mL, where one unit is defined as the amount of enzyme required to liberate 1 mg of maltose from starch in 3 minutes at 20°C, pH 6.9.

Each EnzChek Ultra Amylase Assay Kit provides:

• DQ starch, which is starch from corn that is heavily labeled with the BODIPY FL dye
• Concentrated reaction buffer
• Substrate solvent
• BODIPY FL propionic acid in dimethylsulfoxide (DMSO), for use as a fluorescence standard
• Detailed protocols (EnzChek(R) Ultra Amylase Assay Kit)

This kit provides sufficient reagents for 500 assays using 100 µL assay volumes in a 96-well microplate assay format.

The hydrolysis of xylosidic linkages in hemicellulose polysaccharides by xylanase (EC 3.2.1.8) is important in a wide range of industrial processes, including baking, pulp and paper manufacturing and animal feed production. Xylanases occur in a variety of bacteria and fungi and are classified into two families — glycosyl hydrolase families 10 and 11. Family 11 xylanases are more specific for xylans; family 10 xylanases also exhibit cellulase activity. Existing xylanase assay methods typically require separation or heating steps. The chromogenic substrate o-nitrophenyl-β-D-xylobioside can be used in a simpler homogeneous assay format; however, o-nitrophenol is pH sensitive (pK_a = 7.2) and therefore less than ideal for spectrophotometric assays of xylanases, which exhibit a wide range of pH optima (from pH 2 to 9).

The EnzChek Ultra Xylanase Assay Kit (E33650) provides a quick and convenient mix-and-read assay for measuring xylanase activity or for screening xylanase inhibitors in a high-throughput format using a fluorescence microplate reader or a standard fluorometer (excitation/emission maxima ~358/455 nm). This kit can be used for continuous detection of xylanase activity and offers broad dynamic and pH ranges (1.5 to 200 mU/mL and pH 4–10, respectively), high sensitivity (as low as 1.5 mU/mL) and excellent temperature tolerance. It has been tested with xylanases from Trichoderma viride, T. longibrachiatum, Thermomyces lanuginosus, Aspergillus niger and other bacterial and fungal species. Each EnzChek Ultra Xylanase Assay Kit contains:

• Xylanase substrate
• Concentrated reaction buffer
• Fluorescence standard
• Detailed protocols (EnzChek(R) Ultra Xylanase Assay Kit)

This kit provides sufficient reagents for 500 assays using 100 µL assay volumes in a 96-well microplate assay format.

Lysozyme (muramidase) is an important but difficult enzyme to assay. Lysozyme hydrolyzes β-1-4-glycosidic linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues present in the mucopolysaccharide cell wall of a variety of microorganisms. Lysozyme is present in human serum, urine, tears, seminal fluid and milk. Serum and urine lysozyme levels may be elevated in acute myelomonocytic leukemia (FAB-M4), chronic myelomonocytic leukemia (CMML) and chronic myelocytic leukemia (CML). Increased serum lysozyme activity is also present in tuberculosis, sarcoidosis, megaloblastic anemias, acute bacterial infections, ulcerative colitis and Crohn's disease. Elevated levels of urine and serum lysozyme occur during severe renal insufficiency, renal transplant rejection, urinary tract infections, glomerulonephritis and nephrosis.

Molecular Probes has developed a simple and sensitive assay that can continuously measure the activity of lysozyme in solution. Our fluorescence-based EnzChek Lysozyme Assay Kit (E22013) permits the detection of as little as 30 U/mL of lysozyme (Figure 10.28). One unit of lysozyme is the quantity of enzyme that produces a decrease in turbidity of 0.001 optical density units per minute at 450 nm measured at pH 7.0 (25°C) using a 0.3 mg/mL suspension of Micrococcus lysodeikticus cells as substrate. Our EnzChek lysozyme assay measures lysozyme activity on M. lysodeikticus cell walls that are labeled with fluorescein to such a degree that fluorescence is quenched. Lysozyme action can relieve the quenching, yielding a dramatic increase in fluorescence that is proportional to lysozyme activity. This increase in fluorescence can be measured with any spectrofluorometer, mini-fluorometer or fluorescence microplate reader that can detect fluorescein (excitation/emission maxima ~490/525 nm).

Figure 10.28 Detection of lysozyme activity using the EnzChek Lysozyme Assay Kit (E22013). Increasing amounts of lysozyme were incubated with the DQ lysozyme substrate for 60 minutes at 37°C. The fluorescence was measured in a fluorescence microplate reader using excitation/emission wavelengths of ~485/530 nm. Background fluorescence, determined for a no-enzyme control, was subtracted from each value.

The EnzChek Lysozyme Assay Kit (E22013) contains:

• DQ lysozyme substrate — a fluorescein conjugate of Micrococcus lysodeikticus so heavily labeled that its fluorescence is strongly quenched
• Reaction buffer
• Lysozyme from chicken egg white, for use as a positive control
• Detailed protocols (EnzChek(R) Lysozyme Assay Kit)

Each kit contains sufficient materials for approximately 400 assays of 100 µL in a fluorescence microplate reader.

The widely used β-galactosidase substrate — 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal, B1690, B22015) — yields a dark blue precipitate at the site of enzymatic activity. X-Gal is useful for numerous histochemical and molecular biology applications, including detection of lacZ activity in cells and tissues. In contrast to β-glucuronidase as a gene marker, β-galactosidase can be fixed in cells and tissues with glutaraldehyde without loss of activity and detected with high resolution with X-Gal.

The chromogenic substrate 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid (X-GlcU, B1691) forms a dark blue precipitate. X-GlcU is routinely used to detect GUS expression in transformed plant cells and tissues. However, because it is relatively difficult to differentiate the blue color of the product of X-GlcU against the dark green chloroplasts, we also offer the isomeric 5-bromo-6-chloro-3-indolyl β-D-glucuronide (B8408), which forms a magenta-colored precipitate. X-GlcU can also be used to detect E. coli contamination in food and water.

Phenylethyl β-D-Thiogalactopyranoside (PETG)

Phenylethyl β-D-thiogalactopyranoside (PETG, P1692) is a cell-permeant inhibitor of β-galactosidase activity. We provide PETG in our FluoReporter, DetectaGene Green, ImaGene Green and ImaGene Red lacZ Gene Expression Kits for stopping the enzymatic reaction.

Streptavidin Conjugate of β-Galactosidase

Molecular Probes also offers the streptavidin conjugate of β-galactosidase (S931), a reagent used in a variety of ELISAs. β-D-Galactosidase streptavidin reportedly provided enhanced sensitivity over that obtained with the avidin conjugate of HRP in the detection of a variety of mammalian interleukins and their receptors by ELISA. This reagent has also been used in fluorometric-reverse (IgE-capture) and fluorescence-sandwich ELISAs.

Rabbit Anti–β-Galactosidase Antibody

Molecular Probes offers a polyclonal antibody to the widely used reporter gene product, β-galactosidase. Our rabbit anti–β-galactosidase antibody (A11132) is raised against E. coli–derived β-galactosidase and demonstrates high selectivity for the enzyme. Whether it is being used as a reporter gene or to generate fusion proteins, anti–β-galactosidase provides an easy tool for detecting the enzyme. The antibody is suited to a variety of techniques, including immunoblotting, ELISA, immunoprecipitation and most immunological methods. β-Galactosidase has been used as a tag for quantitative detection of molecules expressed on a cell surface in unfixed, live cells, using anti–β-galactosidase and a β-galactosidase substrate for detection. This novel "cell-ELISA" technique is reported to be applicable to adherent cells and nonadherent cells and to have utility for large-scale screening for expression of cell-surface molecules and of hybridomas for production of antibodies to cell-surface epitopes. This rabbit polyclonal antibody can be used in combination with any of our Zenon Rabbit IgG Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14).

Rabbit Anti–β-Glucuronidase Antibody

In combination with a fluorophore- or enzyme-labeled anti–rabbit IgG secondary antibody (Secondary Immunoreagents - Section 7.2, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1) or any of our Zenon Rabbit IgG Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14), our anti–β-glucuronidase antibody (A5790) can be used to detect the GUS enzyme in transformed plant tissue and in transfected animal cells using Western blotting or immunohistochemical techniques. Furthermore, this antibody, which is raised in rabbits against E. coli–type X-A β-glucuronidase, can be immobilized in microplate wells in order to capture the GUS enzyme from cell lysates. The enzymatic activity can subsequently be determined using any of our fluorogenic or chromogenic β-glucuronidase substrates (see above).

ManLev: A Metabolically Active Carbohydrate Analog

N-Levulinoyl-D-mannosamine (ManLev, L20492; Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2, ) and N-levulinoyl-D-mannosamine, tetraacetate (ManLev tetraacetate, L20493; Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) are ketone-containing monosaccharides that serve as substrates in the oligosaccharide synthesis pathway, resulting in ketone-tagged cell-surface oligosaccharides. Because ketones are rare in cells, reaction with 2 µM biotinylated aldehyde-reactive probe (ARP, A10550; Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2; ) followed by a fluorescent avidin or streptavidin conjugate (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) provides a means of identifying and tracing tagged cells by either imaging () or flow cytometry.

Related Products for Carbohydrate Research

Molecular Probes offers an extensive assortment of reagents for detection and analysis of carbohydrates that are described in other sections of this Handbook. These products include:

• Hydrazine, hydroxylamine and aromatic amine reagents for derivatization and analysis of reducing sugars and periodate-oxidized carbohydrates by electrophoretic methods (Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2, Molecular Probes' hydrazine, hydroxylamine and amine derivatives - Table 3.1)
• Lectins and fluorescent lectin conjugates (Lectins and Other Carbohydrate-Binding Proteins - Section 7.7, Molecular Probes' selection of lectin conjugates - Table 7.24)
• Pro-Q Glycoprotein Blot and Gel Stain Kits (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4)
• Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495, Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2)
• Fluorescent lipopolysaccharides (Sphingolipids, Steroids, Lipopolysaccharides and Related Probes - Section 13.3, Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1; Fluorescent lipopolysaccharide conjugates - Table 16.1)
• Fluorescent glycolipids, including phosphatidylinositol derivatives (Fatty Acid Analogs and Phospholipids - Section 13.2, Sphingolipids, Steroids, Lipopolysaccharides and Related Probes - Section 13.3)
• BODIPY FL C₅-ganglioside G_M1 (B13950, Sphingolipids, Steroids, Lipopolysaccharides and Related Probes - Section 13.3)
• Fluorescent and biotinylated dextrans (Fluorescent and Biotinylated Dextrans - Section 14.5, Molecular Probes' selection of dextran conjugates - Table 14.4)
• NBD-glucosamine derivatives for glucose-transport studies (N13195, N23106; Viability and Cytotoxicity Assay Reagents - Section 15.2)
• Fluorescein insulin (I13269, Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1)
• Fluorescent phosphatidylinositol phosphate derivatives (BODIPY-dye-labeled-phosphoinositides) and antibodies to phosphatidylinositol phosphates (Probes for Lipid Metabolism and Signaling - Section 17.4)