Lectin Analysis of Proteins Blotted onto Filters

Hudson H. Freeze1

1 La Jolla Cancer Research Foundation, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.7
DOI:  10.1002/0471142727.mb1707s23
Online Posting Date:  May, 2001
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Abstract

Lectins are proteins that bind with great specificity to certain carbohydrate structures. Plant lectins are widely used for investigations of carbohydrate structure and for fractionation and purification of individual oligosaccharides and glycopeptides. This unit describes the use of lectins as sensitive indicators for the presence of certain carbohydrate structures linked to proteins blotted onto filters. A tagged lectin is incubated with a blot containing the target protein and binding of the lectin is detected by one of several different procedures. Direct approaches include using lectins labeled with 125I or conjugated to horseradish peroxidase or alkaline phosphatase, which can be detected by chromogenic or luminescent visualization systems. Indirect approaches involve using lectins conjugated to biotin or digoxigenin followed by a second incubation with alkaline phosphatase‐conjugated avidin or antibodies specific for the haptenic digoxigenin group and then by visualization. Several commercial kits are available that provide labeled lectins, control proteins, and developing reagents needed for visualization. These systems can also be adapted for use with lectins other than those supplied with kits. The protocol in this unit is easy to perform with or without a kit. However, the results, while suggestive of carbohydrate structure, are not definitive.

     
 
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Table of Contents

  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Purified protein sample containing 1 to 10 µg of target protein
  • Lectin blot kit (e.g., E‐Y Laboratories Lectin Staining Kit; Genzyme Lectin Link Kit; Boehringer Mannheim Glycan Differentiation Kit) or equivalent materials:
    • Glycoprotein known to bind with the chosen lectins (positive control; Table 17.7.1)
    • Blocking solution: e.g., 1% to 2% (w/v) gelatin in TTBS or 3% periodate‐oxidized bovine serum albumin (BSA)
    • Incubation buffer: e.g., 0.1% (v/v) Tween 20 in Tris‐buffered saline (TTBS; unit 10.8)
    • Labeled or conjugated lectin (Tables 17.7.2 & 17.7.3) in TTBS or other appropriate incubation buffer
    • Enzyme‐ or antibody‐linked antibody (secondary reagent), if needed
    • Chromogenic or luminescent visualization reagent for detecting tagged lectin or antibody (Table 97.80.4711)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2), immunoblotting and immunodetection (unit 10.8), and autoradiography ( appendix 3)
    Table 7.7.1   Materials   Lectin‐Binding Glycoproteins a   Lectin‐Binding GlycoproteinsLectins Suitable for Analysis of Proteins Blotted onto FiltersCommercial Sources of Lectins for Detecting Blotted Glycoproteins

    Glycoprotein Molecular weight (kDa) Lectin bound b
    RNase B 17 Con A
    Ovalbumin 45 Con A
    Transferrin 80 SNA
    Fetuin 68 SNA, MAA
    Asialofetuin 61 RCA I
    α1‐acid glycoprotein 45 DSA
    Lectin Specifity c Notes Kit d
    Con‐A (Concanavalin A) αMan, αGlc Strongly indicates N‐linked oligosaccharide chains. Binds high mannose, hybrid, or bianntennery complex chains EY‐L GN‐LL
    GNA (Galanthus nivalis agglutinin) ManαMan Binds terminal mannose‐linked, α1‐3, α1‐6 or α1‐2 linked to mannose. It will identify “high mannose” N‐glycan chains or O‐glycosidically linked mannose chains in yeast glycoproteins BM‐GD
    WGA (Wheat germ agglutinin) β‐GlcNAc or sialic acid Binds terminal βGlcNAc or sialic acids on various glycans GN‐LL, EY‐L
    RCA I (Ricinus communis agglutinin) βGal Binds Gal‐terminated N‐linked oligosaccharides, but not exclusive GN‐LL
    DSA (Datura stramonium agglutinin) Galβ1‐4GlcNAc Binds Galβ‐1‐4GlcNAc in complex and hybrid N‐glycans and GlcNAc in O‐glycans, also polylactosamines BM‐GD GN‐LL
    MAA (Maackia amurensis agglutinin) SAα 2‐3 Gal‐β‐GlcNAc Binds sialic acid linked α2‐3 to galactose in N‐ and O‐linked glycans BM‐GD GN‐LL
    SNA (Sambucus nigra agglutinin) SAα 2‐6 Gal or GalNAc Binds sialic acid linked α2‐6 to Gal or GalNAc; identifies sialylated O‐glycan, N‐glycan chains BM‐GD GN‐LL
    PNA (Peanut agglutinin) Galβ1‐3 GalNAc Recognizes the core disaccharide galactose β1‐3 N ‐acetylgalactosamine O‐glycosidically linked chains (except of yeast glycoproteins) BM‐GD
    Supplier Conjugate
    Biotin Peroxidase Alkaline phosphatase Digoxigenin Lectin conjugates in kits
    E‐Y Labs + + + Avidin/horseradish peroxidase
    Avidin/alkaline phosphatase
    Boehringer Mannheim + + Anti‐digoxygenin Alkaline phosphatase
    Genzyme + Avidin/alkaline phosphatase
    Sigma + +
    ICN + +

     aGlycoproteins known to bind with the chosen lectins are used as positive controls and as standards for quantitative analysis. They are supplied with some lectin blot kits (e.g., Boehringer Mannheim's) or available separately from Sigma.
     bAbbreviations: Con A, concanavalin A; SNA, Sambucus nigra agglutinin; MAA, Maackia amurensis agglutinin; RCA I, Ricinus communis agglutinin I; DSA, Datura stramonium agglutinin.
    Table 7.7.2   Materials   Lectin‐Binding Glycoproteins a   Lectin‐Binding GlycoproteinsLectins Suitable for Analysis of Proteins Blotted onto FiltersCommercial Sources of Lectins for Detecting Blotted Glycoproteins

    Glycoprotein Molecular weight (kDa) Lectin bound b
    RNase B 17 Con A
    Ovalbumin 45 Con A
    Transferrin 80 SNA
    Fetuin 68 SNA, MAA
    Asialofetuin 61 RCA I
    α1‐acid glycoprotein 45 DSA
    Lectin Specifity c Notes Kit d
    Con‐A (Concanavalin A) αMan, αGlc Strongly indicates N‐linked oligosaccharide chains. Binds high mannose, hybrid, or bianntennery complex chains EY‐L GN‐LL
    GNA (Galanthus nivalis agglutinin) ManαMan Binds terminal mannose‐linked, α1‐3, α1‐6 or α1‐2 linked to mannose. It will identify “high mannose” N‐glycan chains or O‐glycosidically linked mannose chains in yeast glycoproteins BM‐GD
    WGA (Wheat germ agglutinin) β‐GlcNAc or sialic acid Binds terminal βGlcNAc or sialic acids on various glycans GN‐LL, EY‐L
    RCA I (Ricinus communis agglutinin) βGal Binds Gal‐terminated N‐linked oligosaccharides, but not exclusive GN‐LL
    DSA (Datura stramonium agglutinin) Galβ1‐4GlcNAc Binds Galβ‐1‐4GlcNAc in complex and hybrid N‐glycans and GlcNAc in O‐glycans, also polylactosamines BM‐GD GN‐LL
    MAA (Maackia amurensis agglutinin) SAα 2‐3 Gal‐β‐GlcNAc Binds sialic acid linked α2‐3 to galactose in N‐ and O‐linked glycans BM‐GD GN‐LL
    SNA (Sambucus nigra agglutinin) SAα 2‐6 Gal or GalNAc Binds sialic acid linked α2‐6 to Gal or GalNAc; identifies sialylated O‐glycan, N‐glycan chains BM‐GD GN‐LL
    PNA (Peanut agglutinin) Galβ1‐3 GalNAc Recognizes the core disaccharide galactose β1‐3 N ‐acetylgalactosamine O‐glycosidically linked chains (except of yeast glycoproteins) BM‐GD
    Supplier Conjugate
    Biotin Peroxidase Alkaline phosphatase Digoxigenin Lectin conjugates in kits
    E‐Y Labs + + + Avidin/horseradish peroxidase
    Avidin/alkaline phosphatase
    Boehringer Mannheim + + Anti‐digoxygenin Alkaline phosphatase
    Genzyme + Avidin/alkaline phosphatase
    Sigma + +
    ICN + +

     cAbbreviations: Man, mannose; Glc, glucose; GlcNAc, N‐acetylglucosamine; Gal, galactose; SA, sialic acid; GalNAc, N‐acetylgalactosamine.
     dEY‐L, E‐Y Laboratories Lectin Staining Kit; BM‐GD, Boehringer Mannheim Glycan Differentiation Kit; GN‐LL, Genzyme Lectin Link Kit.
    Table 7.7.3   Materials   Lectin‐Binding Glycoproteins a   Lectin‐Binding GlycoproteinsLectins Suitable for Analysis of Proteins Blotted onto FiltersCommercial Sources of Lectins for Detecting Blotted Glycoproteins

    Glycoprotein Molecular weight (kDa) Lectin bound b
    RNase B 17 Con A
    Ovalbumin 45 Con A
    Transferrin 80 SNA
    Fetuin 68 SNA, MAA
    Asialofetuin 61 RCA I
    α1‐acid glycoprotein 45 DSA
    Lectin Specifity c Notes Kit d
    Con‐A (Concanavalin A) αMan, αGlc Strongly indicates N‐linked oligosaccharide chains. Binds high mannose, hybrid, or bianntennery complex chains EY‐L GN‐LL
    GNA (Galanthus nivalis agglutinin) ManαMan Binds terminal mannose‐linked, α1‐3, α1‐6 or α1‐2 linked to mannose. It will identify “high mannose” N‐glycan chains or O‐glycosidically linked mannose chains in yeast glycoproteins BM‐GD
    WGA (Wheat germ agglutinin) β‐GlcNAc or sialic acid Binds terminal βGlcNAc or sialic acids on various glycans GN‐LL, EY‐L
    RCA I (Ricinus communis agglutinin) βGal Binds Gal‐terminated N‐linked oligosaccharides, but not exclusive GN‐LL
    DSA (Datura stramonium agglutinin) Galβ1‐4GlcNAc Binds Galβ‐1‐4GlcNAc in complex and hybrid N‐glycans and GlcNAc in O‐glycans, also polylactosamines BM‐GD GN‐LL
    MAA (Maackia amurensis agglutinin) SAα 2‐3 Gal‐β‐GlcNAc Binds sialic acid linked α2‐3 to galactose in N‐ and O‐linked glycans BM‐GD GN‐LL
    SNA (Sambucus nigra agglutinin) SAα 2‐6 Gal or GalNAc Binds sialic acid linked α2‐6 to Gal or GalNAc; identifies sialylated O‐glycan, N‐glycan chains BM‐GD GN‐LL
    PNA (Peanut agglutinin) Galβ1‐3 GalNAc Recognizes the core disaccharide galactose β1‐3 N ‐acetylgalactosamine O‐glycosidically linked chains (except of yeast glycoproteins) BM‐GD
    Supplier Conjugate
    Biotin Peroxidase Alkaline phosphatase Digoxigenin Lectin conjugates in kits
    E‐Y Labs + + + Avidin/horseradish peroxidase
    Avidin/alkaline phosphatase
    Boehringer Mannheim + + Anti‐digoxygenin Alkaline phosphatase
    Genzyme + Avidin/alkaline phosphatase
    Sigma + +
    ICN + +

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Figures

Videos

Literature Cited

Literature Cited
   Glass, W.F., Briggs, R.C., and Hnilica, L.S. 1981. Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose. Anal. Biochem. 115:219‐224.
  Detailed references regarding detection of the various types of conjugates are given in UNIT . Kit manufacturers will supply relevant references upon request. Much of their information may have been obtained exclusively from data produced in company laboratories, although some has also appeared in the peer‐reviewed literature.
   Sata, T., Zuber, C., and Roth, J. 1990. Lectin‐digoxigenin conjugates: A new hapten system for glycoconjugate cytochemistry. Histochem. 94:1‐11.
   West, C.M. and McMahon, D. 1977. Identification of concanavalin A receptors and galactose‐binding proteins in purified plasma membranes of Dictyostelium discoideum. J. Cell Biol. 74:264‐273.
Key References
   Haselbeck, A., Schickaneder, E., von der Eltz, H., and Hösel, W. 1990. Structural characterization of glycoprotein carbohydrate chains by using digoxigenin‐labeled lectins on blots. Anal. Biochem. 191:25‐30.
  Description of techniques for digoxigenin‐labeled lectins, showing uses for highly purified glycoproteins on blots.
   Tanner, M.J. and Anstee, D.J. 1976. A method for the direct demonstration of the lectin‐binding components of the human erythrocyte membrane. Biochem. J. 153:265‐270.
  Good overview of procedure.
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