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Wheat Germ Agglutinin (WGA)

Wheat germ agglutinin (WGA) is a carbohydrate-binding lectin originally derived from Triticum vulgare. Like other lectins, such as Concanavalin A (ConA), WGA exhibits a high binding affinity for distinct sugar residues commonly present on cell-surface and intracellular glycoconjugates (e.g., glycoproteins and glycolipids). This specificity is often exploited in fluorescence imaging and analysis techniques to label the plasma membranes of eukaryotic cells, gram-positive bacteria, and yeast bud scars, stain fibrotic scar tissue, and localize and detect glycosylated proteins in gels. Fluorescent WGA conjugates, including those labeled with bright, photostable iFluor® dyes, provide greater flexibility in designing colocalization and other multicolor experiments for imaging and flow cytometry.

Properties of Wheat Germ Agglutinin

Wheat germ agglutinin selectively binds to N-acetylglucosaminyl (GlcNAc) and N-acetylneuraminic acid (sialic acid) residues on glycoconjugates and oligosaccharides found throughout the cell surface. The presence and distribution of these residues can not only vary among cell and organelle types but can also be expressed abnormally in various pathologies, making WGA a useful marker in identifying and profiling these phenotypes. WGA is water-soluble and, in solution, exists primarily as a heterodimer with a molecular weight of ∼38 kDa. Under physiological conditions, WGA is cationic (pl > 9) and unable to penetrate live cells. Cell and tissue samples must be fixed before staining with fluorescent WGA conjugates to detect intracellular glycoconjugates and oligosaccharides.

When succinylated with succinic anhydride, the solubility of WGA increases 100-fold at neutral pH and can agglutinate rabbit and human erythrocytes (A, B, and O blood types) with a minimum concentration of 2 µg/mL. Like unmodified WGA, succinylated WGA is specific for GlcNAc residues. However, it loses its binding affinity for sialic acid residues suggesting the use of both lectins as a system for distinguishing between N-acetylglucosaminylated and sialylated glycoconjugates.

Fluorescent WGA conjugates are commonly used in imaging and flow cytometry to localize and detect cell-surface or intracellular glycoconjugates. They are useful at identifying cell types based on glycoprotein-makeup and for labeling eukaryotic cell walls, fibrotic scar tissue, yeast bud scars, chitin and bacterial cell wall peptidoglycans. The latter has been used to differentiate gram-positive and gram-negative bacteria.

Fluorescent iFluor®-WGA Conjugates

AAT Bioquest offers iFluor®-WGA conjugates in multiple fluorescence colors ranging from deep blue through NIR wavelengths, in particular our green-fluorescent iFluor® 488, red-fluorescent iFluor® 594, and far-red fluorescent iFluor® 647-WGA conjugates. These conjugates have nearly identical spectra to their respective FITC, Texas Red, and Cy5 counterparts, but provide significantly brighter more photostable fluorescence signals over a broader pH range (pH 4 to 10). The longer-wavelength iFluor® 647-WGA conjugate, which emits maximally at 670 nm, offers significant advantages in multicolor imaging or when working with samples known to have high intrinsic autofluorescence, such as tissues. The fluorescence emission of iFluor® 647 is well-separated from that of other commonly used blue- and green-fluorescent dyes, eliminating the need for compensation.

Also available are several WGA conjugates labeled with our Alexa Fluor® XFD equivalents, including the widely-used Alexa Fluor® 488 and Alexa Fluor® 594 dyes, among others. All of the XFD dyes have the exact same chemical structure as Alexa Fluor® alternatives.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate stock solution (200X)

Add 500 µL of ddH2O into the powder form to make a 2 mg/mL stock solution.

Note: The reconstituted conjugate solution can be stored at 2-8 °C for short-term storage or at -20 °C for long-term storage.

PREPARATION OF WORKING SOLUTION

iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate working solution (1X)

Add 5 µL of 200X WGA conjugate solution to 1 mL HHBS Buffer.

Note: The optimized staining concentration may be different with different cell lines. The recommended starting concentration is 5-10 µg/mL for live cells.

SAMPLE EXPERIMENTAL PROTOCOL

Warm the vial to room temperature centrifuge briefly before opening. Staining protocols vary with applications. Appropriate dilution of conjugates should be determined experimentally.

Live Cells Stain
  1. Wash cells twice with a HHBS buffer.
  2. Add 100 µL iFluor® 488-WGA working solution.

  3. Incubate cells with WGA working solution for 10-30 minutes at 37 °C.
  4. Wash cells twice with HHBS buffer.
  5. Image cells on a fluorescence microscope using FITC filter set.
Fixed Cells Stain

WGA conjugates can be also used to stain fixed cells.

  1. Fix cells with 4% Formaldehyde in PBS.

    Note: For fixed cell membrane staining, it is recommended to stain without the permeabilization step. A permeabilization step after fixation can facilitate staining intracellular compartments such as Golgi and Endoplasmic Reticulum (ER) structures. 

  2. Add 100 µL iFluor® 488-WGA working solution.

  3. Incubate cells with WGA working solution for 10-30 minutes at room temperature.
  4. Wash cells twice with HHBS buffer.
  5. Image cells on a fluorescence microscope using FITC filter set.

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 555-Wheat Germ Agglutinin (WGA) Conjugate55757010000010.6410.230.14
iFluor® 594-Wheat Germ Agglutinin (WGA) Conjugate58760320000010.5310.050.04
iFluor® 647-Wheat Germ Agglutinin (WGA) Conjugate65667025000010.2510.030.03
iFluor® 350-Wheat Germ Agglutinin (WGA) Conjugate3454502000010.9510.830.23
iFluor® 532-Wheat Germ Agglutinin (WGA) Conjugate5375609000010.6810.260.16
iFluor® 680-Wheat Germ Agglutinin (WGA) Conjugate68470122000010.2310.0970.094
iFluor® 700-Wheat Germ Agglutinin (WGA) Conjugate69071322000010.2310.090.04
iFluor® 750-Wheat Germ Agglutinin (WGA) Conjugate75777927500010.1210.0440.039
iFluor® 790-Wheat Germ Agglutinin (WGA) Conjugate78781225000010.1310.10.09

Citations

View all 10 citations: Citation Explorer
Regulation of H9C2 cell hypertrophy by 14-3-3$\eta$ via inhibiting glycolysis
Authors: Wan, Sha and Wang, Songhao and Yang, Xianfei and Cui, Yalan and Guan, Heng and Xiao, Wenping and Liu, Fang
Journal: PloS one (2024): e0307696
A novel function of claudin-5 in maintaining the structural integrity of the heart and its implications in cardiac pathology
Authors: Zhang, Yi and Chen, Baihe and Wang, Miao and Liu, Haiqiong and Chen, Minjun and Zhu, Jiabiao and Zhang, Yu and Wang, Xianbao and Wu, Yuanzhou and Liu, Daishun and others,
Journal: Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease (2024): 167274
USP7 Promotes Cardiometabolic Disorders and Mitochondrial Homeostasis Dysfunction in Diabetic Mice via stabilizing PGC1$\beta$
Authors: Yan, Meiling and Su, Liyan and Wu, Kaile and Mei, Yu and Liu, Zhou and Chen, Yifan and Zeng, Wenru and Xiao, Yang and Zhang, Jingfei and Cai, Guida and others,
Journal: Pharmacological Research (2024): 107235
PDE5 inhibition mitigates heart failure in hyperlipidemia
Authors: Huang, Wuqing and Yang, Xi and Zhang, Naiqi and Chen, Keyuan and Xiao, Jun and Qiu, Zhihuang and You, Sujun and Gao, Ziting and Ji, Jianguang and Chen, Liangwan
Journal: Biomedicine \& Pharmacotherapy (2024): 116710

References

View all 50 references: Citation Explorer
Wheat germ agglutinin liposomes with surface grafted cyclodextrins as bioadhesive dual-drug delivery nanocarriers to treat oral cells.
Authors: Wijetunge, Sashini S and Wen, Jianchuan and Yeh, Chih-Ko and Sun, Yuyu
Journal: Colloids and surfaces. B, Biointerfaces (2020): 110572
Wheat germ agglutinin-conjugated fluorescent pH sensors for visualizing proton fluxes.
Authors: Zhang, Lejie and Zhang, Mei and Bellve, Karl and Fogarty, Kevin E and Castro, Maite A and Brauchi, Sebastian and Kobertz, William R
Journal: The Journal of general physiology (2020)
Wheat germ agglutinin is a biomarker of whole grain content in wheat flour and pasta.
Authors: Killilea, David W and McQueen, Rebecca and Abegania, Judi R
Journal: Journal of food science (2020): 808-815
Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of concanavalin A- and wheat germ agglutinin-reactive glycans mark seminal prostasome populations from normozoospermic and oligozoospermic men.
Authors: Janković, Tamara and Goč, Sanja and Mitić, Ninoslav and Danilović Luković, Jelena and Janković, Miroslava
Journal: Upsala journal of medical sciences (2020): 10-18
Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1.
Authors: Guevara, Rebekah B and Fox, Barbara A and Bzik, David J
Journal: mSphere (2020)
Page updated on October 8, 2024

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Physical properties

Solvent

Water

Spectral properties

Correction Factor (260 nm)

0.21

Correction Factor (280 nm)

0.11

Extinction coefficient (cm -1 M -1)

750001

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.91

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

Platform

Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall, clear bottom
Live HeLa cells were stained with&nbsp;iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate at 5&nbsp;&micro;g/mL for 30 minutes followed by Hoechst 33342 (AAT Cat# 17535). Image was acquired using fluorescence microscopy using FITC and DAPI filter set.
Live HeLa cells were stained with&nbsp;iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate at 5&nbsp;&micro;g/mL for 30 minutes followed by Hoechst 33342 (AAT Cat# 17535). Image was acquired using fluorescence microscopy using FITC and DAPI filter set.
Live HeLa cells were stained with&nbsp;iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate at 5&nbsp;&micro;g/mL for 30 minutes followed by Hoechst 33342 (AAT Cat# 17535). Image was acquired using fluorescence microscopy using FITC and DAPI filter set.