Cellular proteoglycans, glycoproteins and glycolipids may contain any of a wide variety of oligosaccharides. Although most abundant on the cell surface, oligosaccharide residues are sometimes also found covalently attached to constituents within the cell. Often, specific oligosaccharides are associated with a certain cell type or organelle. Lectins and certain other carbohydrate-binding proteins that bind to specific configurations of sugar molecules can thus serve to identify cell types or cellular components, making them versatile primary detection reagents in histochemical applications and flow cytometry (, , ). Fluorescent derivatives of carbohydrate-binding proteins and other reagents have been used to detect cell-surface and intracellular glycoconjugates by microscopy and flow cytometry, to localize glycoproteins in gels and on protein blots, to precipitate glycoproteins from solution and to cause agglutination of specific cell types. In addition, lectins are also useful markers of certain cancers because these cells often display altered surface glycoproteins.
Properties of Concanavalin A and Wheat Germ Agglutinin
Concanavalin A (Con A) selectively binds to α-mannopyranosyl and α-glucopyranosyl residues. In moderately acidic solutions (pH 4.5–5.6), Con A exists as a dimer with a molecular weight of approximately 52,000 daltons; above pH 7, it is primarily a tetramer with a molecular weight of 104,000 daltons. Con A is a metalloprotein that requires one Ca2+ and one Mn2+ per subunit for carbohydrate binding. The 36,000-dalton dimeric wheat germ agglutinin (WGA), which is normally cationic, binds to N-acetylneuraminic acid (sialic acid) and N-acetylglucosaminyl residues.
When Con A is succinylated with succinic anhydride, as in the Alexa Fluor 488 conjugate of succinylated Con A (C21401), it is irreversibly converted to a dimer that retains the same sugar-binding specificity as the parent lectin. Succinylated Con A, however, has a profile of biological activities quite different from that of the tetrameric form. In contrast to the tetramer, succinylated Con A does not induce capping of cell-surface glycoprotein receptors, nor does it inhibit capping of cell-surface immunoglobulin receptors or strongly agglutinate erythrocytes or spleen cells. The mitogenic effect of succinylated Con A is similar to that of the native lectin, although it is mitogenic over a significantly wider range of concentrations than is the tetramer.
Con A and WGA Conjugates
We offer several fluorescent conjugates of Con A and WGA—two of the most commonly used lectins in cell biology (Molecular Probes lectin conjugates—Table 7.10). Although we continue to provide fluorescein and tetramethylrhodamine conjugates of these lectins, we strongly recommend that researchers also evaluate our other fluorescent conjugates, which exhibit excitation and emission maxima nearly identical to these traditional reagents but exhibit superior brightness and photostability. In particular, our green-fluorescent Alexa Fluor 488 conjugates of Con A, succinylated Con A and WGA (C11252, C21401, W11261) are not only brighter than fluorescein conjugates, but also much more photostable and pH insensitive in the physiological range (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3). Moreover, the red-fluorescent Alexa Fluor 594 conjugates of Con A and WGA (C11253, W11262) are even more fluorescent than their Texas Red counterparts. The Alexa Fluor 633 and Alexa Fluor 647 conjugates of Con A (C21402, C21421) and WGA (W21404, W32466) and the Alexa Fluor 680 conjugate of WGA (W32465) emit light at long wavelengths and are exceptionally useful for multicolor fluorescent labeling of cells and tissues, including those that have high intrinsic autofluorescence. For blue-fluorescent labeling, we recommend the Alexa Fluor 350 conjugates of Con A and WGA (C11254, W11263; ). For researchers interested in testing our fluorescent WGA conjugates in their various applications, we offer a Wheat Germ Agglutinin Sampler Kit (W7024), which contains 1 mg quantities each of WGA conjugates of the Alexa Fluor 350, Oregon Green 488, tetramethylrhodamine and Texas Red-X dyes.
The Qdot 655 WGA conjugate (Q12021MP) extends our selection of dye-labeled WGA conjugates for labeling of N-acetylglucosaminyl and sialic acid residues of glycoproteins on cell surfaces; the physical and spectroscopic properties of Qdot nanocrystals are described in Qdot Nanocrystals—Section 6.6.
Applications for Fluorescent Con A and WGA
Although the distribution of oligosaccharides that may be bound by Con A and WGA varies widely among cell types, these two lectins have proven to be useful reagents for a number of applications, including immunolocalization of oncogene products, specific intracellular enzymes, viral proteins and components of the cytoskeleton (). Con A also reportedly binds specifically to isolated Golgi fractions from rat liver, which enabled researchers to use fluorescein-labeled Con A to examine the effect of chronic ethanol intake on carbohydrate content in these organelles using flow cytometry. Fluorescent Con A has been used to:
- Determine if human sperm cells have undergone the progesterone-induced acrosome reaction
- Investigate receptor capping in leukocytes
- Measure lateral diffusion of glycoproteins, glycolipids and viruses in membranes
- Show the redistribution of cell-surface glycoproteins in murine fibroblasts that had been induced to migrate by exposure to an electric field
WGA conjugates are useful for labeling of bacterial cell wall peptidoglycans, chitin and cartilage glycosaminoglycans. In addition, nuclear core complexes have been found to contain several proteins with O-linked N-acetylglucosaminyl residues. In a study of nuclear protein transport, nuclei isolated from monkey kidney epithelial cells were demonstrated to be intact by their bright staining with fluorescein WGA (W834); fluorescein Con A (C827), which binds to residues accessible only in nuclei with compromised membranes, was used as a negative control for intact nuclei. Fluorescent WGA has also been employed to monitor reconstitution of the nuclear core complex in Xenopus egg extracts. WGA conjugates undergo axonal transport and have been shown to cross from axonal nerve endings into adjacent neurons.
Fluorescent lectins are also useful in microbiology applications. Fluorescent WGA conjugates stain chitin in fungal cell walls and have been reported to stain gram-positive but not gram-negative bacteria for subsequent analysis by either imaging or flow cytometry. Fluorescent WGA conjugates are utilized in our ViaGram Red+ Bacterial Gram Stain and Viability Kit (V7023, Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) to differentiate gram-positive and gram-negative bacteria. Fluorescent WGA also binds to sheathed microfilariae and has been used to detect filarial infection in blood smears.
In addition to these nuclear core and microbiology studies, fluorescent WGA has been used to:
- Bind the sarcolemma of rat and dog cardiac myocytes, even within the intercalated discs and transverse tubules, allowing researchers to map the distribution of gap junctions in these cell types
- Determine the intracellular distribution of altered lysosomal proteins, enabling the definition of the sequence requirements for proper cell sorting
- Identify the differentiation state of Madin–Darby canine kidney (MDCK) cells
- Investigate plant hemicelluloses
- Measure cell membrane potential in combination with potential-sensitive membrane probes
Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit
The Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit (I34406) provides two stains—red-fluorescent Alexa Fluor 594 wheat germ agglutinin (WGA) (excitation/emission maxima ~590/617 nm) and blue-fluorescent Hoechst 33342 dye (excitation/emission maxima when bound to DNA ~350/461 nm)—for highly selective staining of the plasma membrane and nucleus, respectively, of live cells expressing Green Fluorescent Protein (GFP, ). These dyes can be combined into one staining solution using the protocol provided, saving labeling time and wash steps while still providing optimal staining. Cell-impermeant Alexa Fluor 594 WGA binds selectively to N-acetylglucosamine and N-acetylneuraminic acid residues. When used according to the protocol, Alexa Fluor 594 WGA provides highly selective labeling of the plasma membrane with minimal background, although labeling may not be as distinct for flat cell types when viewed using standard epifluorescence microscopy or low magnification. Alexa Fluor 594 WGA is retained after formaldehyde fixation and permeabilization with Triton X-100. This fluorescent lectin conjugate can also be used to label fixed cells; however, to avoid labeling intracellular components, formaldehyde-fixed cells should not be permeabilized before labeling. It is important to note that Alexa Fluor 594 WGA can stimulate biological activity, including clustering of glycosylated cell-surface proteins.
The kit also includes Hoechst 33342 dye, a cell-permeant nucleic acid stain that is selective for DNA and is spectrally similar to DAPI. This dye should not interfere with GFP fluorescence and is retained after fixation and permeabilization. The Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit contains:
- Alexa Fluor 594 wheat germ agglutinin (WGA)
- Hoechst 33342
- Detailed experimental protocols (Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit)
Each kit provides enough staining solution for 360 assays using the protocol provided for labeling live, cultured cells that are adhering to coverslips.
Lectins from Griffonia simplicifolia
Isolectin GS-IB4 is a 114,000-dalton glycoprotein that is part of a family of five tetrameric type I isolectins (IA4, IA3B, IA2B2, IAB3 and IB4) isolated from the seeds of the tropical African legume Griffonia simplicifolia (formerly Bandeiraea simplicifolia). The A and B subunits are very similar, differing in amino acid sequence only at the N-terminus. The subunits, however, display remarkably different binding specificities. The A subunit prefers N-acetyl-D-galactosamine end groups, whereas the B subunit is selective for terminal α-D-galactosyl residues.
We offer Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 647 dye conjugates of this versatile isolectin (I21411, I21412, I21413, I32450), as well as its biotinylated (I21414) derivative. Isolectin GS-IB4 specifically agglutinates blood group B erythrocytes and was originally employed for this purpose. Subsequent work has shown that isolectin GS-IB4 is cytotoxic to several normal and tumor cell types and has particularly strong affinity for brain microglial and perivascular cells (). Conjugates of isolectin GS-IB4 have been particularly valuable as histochemical and flow cytometric probes for specifically labeling endothelial cells in a number of species. They have also been used effectively for tracing central and peripheral neuronal pathways following local injections.
Lectin GS-II from G. simplicifolia differs from isolectin S-IB4 in that it is the only known lectin that binds with high selectivity to terminal, nonreducing α- and β-N-acetyl-D-glucosaminyl (GlcNAc) residues of glycoproteins. Lectin GS-II is a tetrameric protein with each site binding a single carbohydrate and an aggregate molecular weight of ~113,000 daltons. Because of its affinity for GlcNAc, lectin GS-II conjugates are useful for staining intermediate-to-trans Golgi—the site of N-acetylglucosaminyltransferase activity. The Golgi apparatus of oligodendrocytes and ganglion neurons are also stained by fluorescent GS-II conjugates. We have prepared the green-fluorescent Alexa Fluor 488 (L21415, ), red-fluorescent Alexa Fluor 594 (L21416) and far-red–fluorescent Alexa Fluor 647 (L32451) conjugates of lectin GS-II for use in cell staining.
Phaseolus vulgaris (Red Kidney Bean) Lectin
Phaseolus vulgaris lectin (PHA-L) is a tetrameric protein with a molecular weight of 120,000 daltons. Its binding to glycoproteins is strongly inhibited by N-acetylglucosaminyl (1-2) mannopyranosyl residues. Like WGA, PHA-L is widely used as an injectable neuronal tracer. Iontophoretically injected PHA-L clearly demonstrates the morphological features of the filled neurons at the injection site and the labeled axons and axon terminals. Furthermore, PHA-L that has been transported is not degraded over long periods. PHA-L has been used in combination with our biotinylated lysine-fixable dextrans (BDA dextrans; Fluorescent and Biotinylated Dextrans—Section 14.5, Molecular Probes dextran conjugates—Table 14.4) and the fluorescent dextrans fluoro-ruby and mini-ruby (D1817, D3312; Fluorescent and Biotinylated Dextrans—Section 14.5) to demonstrate their similar transport properties and to show the distribution of anterograde-labeled fibers. We have prepared Alexa Fluor 488, Alexa Fluor 594 and Alexa Fluor 647 conjugates of PHA-L (L11270, L32456, L32457).
Arachis hypogaea (Peanut) Lectin
Arachis hypogaea lectin (PNA) is a tetrameric protein with a molecular weight of ~110,000 daltons that has specificity for terminal β-galactose residues of glycoproteins. Lactose strongly inhibits binding of PNA to these glycoproteins, with D-galactose somewhat less effective. PNA binds to a broad range of receptors in human tissues, with some preference for glycoproteins that have been treated with neuraminidase (sialidase) to remove terminal sialic acids. We offer Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 647 conjugates of PNA (L21409, L32458, L32459, L32460).
PNA-binding sites are widespread in human tissues, with staining patterns varying by tissue type. Research has shown PNA to be selective for acrosomes in rat and human sperm, and PNA serves as a marker for certain melanomas. PNA has also been used to label the synaptic extracellular matrix in the study of developing neuromuscular junctions.
Applications for fluorescent PNA conjugates include:
- Binding to desialylated CD44, the PNA receptor in keratinocytes, as a marker of terminal differentiation
- Detecting the acrosome reaction of sperm
- Labeling of photoreceptors in chick embryos and in intact retina and dissociated retinal cells
- Staining colonic mucins in cultured human tumor cells, transitional cell carcinomas in the bladder and leukemic cells
Helix pomatia (Edible Snail) Agglutinin
Helix pomatia agglutinin (HPA), a hexameric protein with a molecular weight of 70,000 daltons, selectively binds to type A erythrocytes and α-N-acetylgalactosaminyl residues (). In some cell types, HPA conjugates may be used as markers for the Golgi apparatus. Furthermore, HPA conjugates have been shown to be as effective as monoclonal antibodies for the detection and differentiation of herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) in cultured cells. Our Alexa Fluor 488 and Alexa Fluor 647 conjugates of HPA (L11271, L32454) should be particularly useful for cell staining.
Glycine max (Soybean) Agglutinin
Soybean agglutinin (SBA) from Glycine max is a tetrameric protein that consists of a mixture of isolectins that have an aggregate molecular weight of ~120,000 daltons. SBA has a stronger reaction with type A1 blood cells than with type A2 blood cells and is known to selectively bind to terminal α- and β-N-acetylgalactosaminyl and galactopyranosyl residues of glycoproteins. Neuraminidase-treated cells react more strongly with SBA conjugates than do untreated cells. SBA has selective affinity for human CD34+ hematopoietic stem cells and lymphocytes, and its immobilized conjugates are important reagents for the depletion of T cells in bone marrow transplantation. In addition, a fluorescein conjugate of SBA was shown to be useful for assessing the stage of lymphoid cell differentiation in human leukemic–lymphoma cell lines. Like many lectins, fluorescent SBA conjugates have been shown to bind with high affinity to several types of tumor cells. SBA conjugates are also reported to be selective stains for glial cells and the zona pellucida of the mammalian egg. For detecting SBA-binding cells, we have prepared the Alexa Fluor 488, Alexa Fluor 594 and Alexa Fluor 647 conjugates of SBA (L11272, L32462, L32463).
Cholera Toxin Subunit B
Cholera toxin comprises two subunits, A and B, arranged in an AB5 conformation. Subunit A is an ADP-ribosyltransferase, which disrupts the proper signaling of G proteins and eventually leads to dehydration of the cell. The 12,000-dalton nontoxic subunit B ("choleragenoid"), which is assembled into a 60,000-dalton pentamer above pH 2, allows the protein complex to bind to cellular surfaces via the pentasaccharide chain of ganglioside GM1.
Fluorescent cholera toxin subunit B, which binds to galactosyl moieties, is a marker of lipid rafts—regions of cell membranes high in ganglioside GM1 that are thought to be important in cell signaling. Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane. Lipid rafts also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and receptors, which may be regulated by their selective interactions with these membrane microdomains. Research has demonstrated that lipid rafts play a role in a variety of cellular processes—including the compartmentalization of cell-signaling events, the regulation of apoptosis and the intracellular trafficking of certain membrane proteins and lipids —as well as in the infectious cycles of several viruses and bacterial pathogens.
The Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405; Tracers for Membrane Labeling—Section 14.4) provide the key reagents for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor dyes (). Live cells are first labeled with the green-fluorescent Alexa Fluor 488, orange-fluorescent Alexa Fluor 555 or red-fluorescent Alexa Fluor 594 conjugate of cholera toxin subunit B (CT-B). This CT-B conjugate binds to the pentasaccharide chain of plasma membrane ganglioside GM1, which selectively partitions into lipid rafts. An antibody that specifically recognizes CT-B is then used to crosslink the CT-B–labeled lipid rafts into distinct patches on the plasma membrane, which are easily visualized by fluorescence microscopy. Each Vybrant Lipid Raft Labeling Kit contains sufficient reagents to label 50 live-cell samples, including:
- Recombinant cholera toxin subunit B (CT-B) labeled with the Alexa Fluor 488 (in Kit V34403), Alexa Fluor 555 (in Kit V34404) or Alexa Fluor 594 (in Kit V34405) dye
- Anti–cholera toxin subunit B antibody (anti–CT-B)
- Concentrated phosphate-buffered saline (PBS)
- Detailed labeling protocol (Vybrant Lipid Raft Labeling Kits)
Cholera toxin subunit B and its conjugates are also established as superior tracers for retrograde labeling of neurons. Cholera toxin subunit B conjugates bind to the pentasaccharide chain of ganglioside GM1 on neuronal cell surfaces and are actively taken up and transported; alternatively, they can be injected by iontophoresis. Unlike the carbocyanine-based neuronal tracers such as DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4), cholera toxin subunit B conjugates can be used on tissue sections that will be fixed and frozen.
All of our cholera toxin subunit B conjugates are prepared from recombinant cholera toxin subunit B, which is completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity. The Alexa Fluor 488 (C22841, C34775), Alexa Fluor 555 (C22843, C34776), Alexa Fluor 594 (C22842, C34777) and Alexa Fluor 647 (C34778) conjugates of cholera toxin subunit B combine this versatile tracer with the superior brightness of our Alexa Fluor dyes to provide sensitive and selective receptor labeling and neuronal tracing. We also offer biotin-XX (C34779) and horseradish peroxidase (C34780) conjugates of cholera toxin subunit B for use in combination with diaminobenzidine (DAB) oxidation, tyramide signal amplification (TSA) and Qdot nanocrystal–streptavidin conjugates.