Imaging with Lectin Fluorescent Conjugates - US (2024)

Imaging with Lectin Fluorescent Conjugates - US (1)

Fluorescent lectins have been used to localize and detect cell-surface or intracellular glycoconjugates, making them versatile primary detection reagents for microscopy or with flow cytometry. Lectins are available conjugated to a wide variety of fluorescent dyes, including bright, photostable Alexa Fluor dyes, increasing the flexibility in designing multicolor experiments for imaging and flow cytometry. Special conjugation procedures are used to optimize the number of dye molecules bound to the lectins, leaving a bright signal while preserving the binding specificity of the individual lectin. High signal-to-noise ratio is also maintained by the removal of unconjugated fluorochromes and inactive lectins.

See lectins selection guide

On this page:

  • Lectin introduction
  • Lectins and their properties selection guide
  • Fluorescent lectin imaging examples

Lectin introduction

Lectins are carbohydrate binding proteins that are highly specific tooligosaccharides, or sugar groups attached to other molecules. They can cause agglutination of specific cells or precipitation of glycoconjugates. Lectins are involved in regulation of cell adhesion, glycoprotein synthesis, and blood protein levels along with playing a role in the immune system.

Oligosaccharides are found throughout the cell surface but can also be found attached within the cell. Specific oligosaccharides and their distribution can vary among cell type and organelle. Lectins, because of their specificity to their target, can serve to identify cell types or cellular components when used in conjunction with various labeling approaches. Fluorescent derivatives, or conjugated lectins, have been used to localize and detect cell-surface or intracellular glycoconjugates, making them versatile primary detection reagents for microscopy or with flow cytometry.Additionally, cellular differentiation of cancerous cell types are often identified by the makeup of their surface glycoproteins, making lectins useful markers in detecting those distinct cellular phenotypes.

Lectins and their properties selection guide

LectinConjugateSourcePreferred sugar specificityBlood group specificityMitogenic activity
Arachis hypogaea (peanut) lectin (PNA)Alexa Fluor 488PeanutT antigen (M, N)ß-gal(1->3)galNAcNo
Alexa Fluor 568
Alexa Fluor 594
Alexa Fluor 647
Concanavalin A (Con A)Alexa Fluor 350Canavalia ensiformis (jack bean)Non-specific after neuraminidase treatmentα-man, α-glcYes
Alexa Fluor 488
Alexa Fluor 594
Alexa Fluor 633
Alexa Fluor 647
Texas-Red
Tetramethylrhodamine (TRITC)
Concanavalin A (ConA), succinylatedAlexa Fluor 488Canavalia ensiformis (jack bean)Non-specific after neuraminidase treatmentα-man, α-glcYes
Dolichos biflorus agglutinin (DBA)Fluorescein (FITC)Horse gramA1α-galNAcNo
Glycine max (soybean) lectin (SBA)Alexa Fluor 594SoybeanA>O>Bα>ßGalNAcYes
Alexa Fluor 647
Griffonia simplicifolia lectin (GSL-II)Alexa Fluor 488Africa shrub legumeB>>A1α or ß GalNAcNo
Alexa Fluor 594
Alexa Fluor 647
Griffonia simplicifoliaisolectin GS-IB4 of B subunits (GS-IB4, GSL-IB4)Alexa Fluor 488Africa shrub legumeBαGalNo
Alexa Fluor 568
Alexa Fluor 594
DyLight 594
Alexa Fluor 647
Biotin-XX conjugate
Helix pomatia lectin (HPA)Alexa Fluor 488Edible snailAgalNAcNo
Alexa Fluor 647
Lens culinaris agglutinin (LCA)Fluorescein (FITC)LentilNon-specific after neuraminidase treatmentαMan, αGlcYes
Lotus tetragonolobus lectin (LTL)Fluorescein (FITC)Asparagus peaO<A2αFucYes
Lycopersicon esculentum lectin (LEL, LEA, TL)DyLight 488TomatoNon-specific after neuraminidase treatment(glcNAc)3Yes
DyLight 594
DyLight 649
Fluorescein (FITC)
Texas Red
Phaseolus vulgaris lectin (PHA-L)Alexa Fluor 488Red kidney beanNon-specific after neuraminidase treatmentGalß4GlcNAcß6(GlcNAc ß2Manα3)Manα3Yes
Alexa Fluor 594
Ricinus communis agglutinin I (RCA I, RCA 120)Fluorescein (FITC)Castor beanNon-specific after neuraminidase treatmentGalNo
Sambucus nigra lectin (SNA, EBL)Fluorescein (FITC)ElderberryNon-specific after neuraminidase treatmentNeu5Acα6Gal/GalNAcNo
Ulex europaeus agglutinin I (UEA I)Fluorescein (FITC)Gorse, furzeo>A2αFucNo
Wheat germ agglutinin (WGA)Alexa Fluor 350Wheat germA, B, OGlcNAcYes
Alexa Fluor 488
Alexa Fluor 555
Alexa Fluor 594
Alexa Fluor 633
Alexa Fluor 647
Alexa Fluor 680
Oregon Green 488
Tetramethylrhodamine
Texas Red-X
Fluorescein
Sampler Kit
Wisteria floribunda lectin (WFA, WFL)Fluorescein (FITC)Japanese wisteriaNon-specific after neuraminidase treatmentGlcNAcNo

Fluorescent lectin imaging examples

Concanavalin A conjugates

Concanavalin A (Con A), a widely used fluorescent lectin marker in cell biology, selectively binds to α‑mannopyranosyl and α‑glucopyranosyl residues (Figure 1). There are several fluorescent markers of Con A and they are useful for various applications including localization of oncogene products, intracellular enzymes,viral proteins,and cytoskeletal components.

Imaging with Lectin Fluorescent Conjugates - US (2)

Figure 1. Osteosarcoma cells stained with Alexa Fluor 488 concanavalin A, Alexa Fluor 594 wheat germ agglutinin, and Hoechst 33342 nucleic acid stain. Fixed and permeabilized osteosarcoma cells simultaneously stained with the fluorescent lectins Alexa Fluor 488 concanavalin A (Con A) and Alexa Fluor 594 wheat germ agglutinin (WGA). Con A selectively binds a-glucopyranosyl residues, whereas WGA selectively binds sialic acid and N-acetylglucosaminyl residues. The nuclei were counterstained with blue fluorescent Hoechst 33342 nucleic acid stain. The image was acquired using bandpass filter sets appropriate for the Texas Red dye, fluorescein, and DAPI.

Wheat germ agglutinin conjugates

Wheat germ agglutinin (WGA), another widely used fluorescent lectin marker in cell biology, selectively binds to N‑acetylglucosamine and N‑acetylneuraminic acid (sialic acid) residues (Figure 2). Similar to Concanavalin A, WGA is useful for localization of oncogene products, specific intracellular enzymes,viral proteins,and cytoskeletal components.

WGA conjugates can also be used as a lectin marker for bacterial cell wall peptidoglycans, chitin, and cartilage glycosaminoglycans. WGA conjugates have been shown to stain gram-positive but not gram-negative bacteria.

Imaging with Lectin Fluorescent Conjugates - US (3)

Figure 2. Multicolor fluorescence image of mouse intestine cross section. Filamentous actin prevalent in the brush border was visualized with Alexa Fluor 568 phalloidin. Goblet cell mucus was detected with a blue-fluorescent Alexa Fluor 350 conjugate of wheat germ agglutinin. Nuclei were stained with SYTOX Green nucleic acid stain.

Arachis hypogaea (peanut) conjugates

Arachis hypogaealectin (PNA), isolated from peanuts, specifically binds terminal β-galactose residues of glycoproteins (Figure 3). After neuraminidase treatment, PNA has been shown to bind human erythrocytes. Since PNA-binding sites are widespread in human tissues, the staining patterns will vary by tissue type.

Imaging with Lectin Fluorescent Conjugates - US (4)

Figure 3. Multicolor fluorescence image of mouse testis cross section. Spermatozoa were detected using the green-fluorescent Alexa Fluor 488 conjugate of lectin PNA. Actin filaments were visualized using red-fluorescent Alexa Fluor 568 phalloidin. Nuclei were stained with blue fluorescent DAPI.

Learn more about fluorescent lectin conjugates

Resources

Molecular Probes Handbook

  • Probes for Cytoskeletal Proteins—Chapter 11
  • Probes for Organelles—Chapter 12
  • Probes for Lipids and Membranes—Chapter 13

BioProbes 70 Journal article

  • Journal Club—An In-Depth Look at Fluorescent Dyes for Organelle Labeling

Research tools

  • Stain-iT™ Cell Staining Simulator
  • Fluorescence SpectraViewer

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