Selective Lectin Stain

For the characterization of glycoproteins with lectins, the sample is run on an SDS gel with an approximate 10-cm-wide pocket and then blotted. The blot (on PVDF membrane) is stained on protein and the approximately 10-cm-long protein band is cut crosswise into approximately 20 narrow strips. After blocking (e.g., with 2% polyvinylpyrrolidon 360 in 50 mM Tris-Cl pH 7.5 and 0.5 M NaCl), the strips are incubated in twos with a labeled lectin (Table 9.1) with or without inhibiting sugar, and are then developed. 

Lectin blots are blocked with substances free from lectin-binding glycoproteins (Tween, polyvinylpyrrolidon 360, purified BSA, periodate-oxidized BSA, hemoglobin). Milk powder and the cheaper BSA preparations contain glycoproteins. A control strip of the blot should be incubated with lectin and competing sugar and show no stain. Don t forget many lectins bind only in the presence of cofactors (e.g., Ca , Mg +, or Mn +). 

If you stain the blots with peroxidase-labeled lectins, you should know that the peroxidase substrate diaminobenzidine is carcinogenic and binds Con A peroxidases. A covalent labeling of Con A is thus unnecessary. Finally, you will have little luck if you transform the lectins with nitrocellulose blots, in that they bind to cellulose (alternative PVDF membranes). Table 9.1 shows a selection of commercially available labeled lectins. 

Hortin, G., and Timpe, B. (1990). Lectin Affinity Chromatography of Proteins Bearing O-linked Oligossac-charides Application of Jacalin-agarose, Anal Biochem. 188 271-277. 

Kijimoto, S., et al. (1985). Analysis of N-linked Oligosaccharide Chains of Glycoproteins on Nitrocellulose Sheets Using Lectin-peroxidase Reagents, Anal. Biochem. 147 222-229. 

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The glomemlar layer of the AOB can be divided into anterior and posterior (rostral and caudal) halves on the basis of their chemoarchitecture (Halpem et al., 1995) the anterior stains more darkly than the posterior when treated with the lectin VVA Vicia villosa agglutinin) (Shapiro et al., 1995), NADPH-diaphorase (Shapiro and Halpem, 1998), and antibodies to olfactory marker protein (OMP) (Shnayder et al., 1993) and the G-protein Gi2-aipha (Halpem et al., 1995). The posterior half is selectively stained by Gq. alpha antibodies (Halpem et al., 1995 Jia and Halpem, 1996). A retrograde HRP labeling study has suggested, and another using immunohistochemical technique has confirmed, that these halves receive projections from distinct laminae of the VNO (Jia and Halpem, 1996 Shapiro et al., 1995). 

In the rat, the monoclonal antibody MEP-1 stained all cells lining the alveolar space, except the type II pneumocytes (Kasper et al. 1996). Double fluorescence staining employing type I cell-specific lectin BPA (Kasper et al. 1994) revealed the type I cell specificity. Immunoelectron microscopy confirmed this selective reaction of the MEP-1 antibody. The polyclonal anti pan-cathedrin antibody selectively decorated type I pneumocytes, alveolar macrophages and endothelial cells of large blood vessels. Caveolin is a selective marker of type I pneumocytes (Kasper and Reimann 1997). 

Plate 21 A fluorescent molecular probe. Fixed and permeabilized osteosarcoma cells were simultaneously stained with the fluorescent lectins, Alexa 488 concanavalin A (Con A) and Alexa 594 wheat germ agglutinin (WGA). Con A selectively binds a-mannopyranosyl and a-glucopyranosyl residues, whereas WGA selectively binds sialic acid counterstained with blue-fluorescent Hoechst 33342 nucleic acid stain. See Fluorescent Molecular Probes, Inc. 

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