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
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
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.
- Wash cells twice with a HHBS buffer.
Add 100 µL iFluor® 488-WGA working solution.
- Incubate cells with WGA working solution for 10-30 minutes at 37 °C.
- Wash cells twice with HHBS buffer.
- Image cells on a fluorescence microscope using FITC filter set.
WGA conjugates can be also used to stain fixed cells.
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.
Add 100 µL iFluor® 488-WGA working solution.
- Incubate cells with WGA working solution for 10-30 minutes at room temperature.
- Wash cells twice with HHBS buffer.
- Image cells on a fluorescence microscope using FITC filter set.
Spectrum
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
iFluor® 555-Wheat Germ Agglutinin (WGA) Conjugate | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
iFluor® 594-Wheat Germ Agglutinin (WGA) Conjugate | 587 | 603 | 2000001 | 0.531 | 0.05 | 0.04 |
iFluor® 647-Wheat Germ Agglutinin (WGA) Conjugate | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
iFluor® 350-Wheat Germ Agglutinin (WGA) Conjugate | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
iFluor® 532-Wheat Germ Agglutinin (WGA) Conjugate | 537 | 560 | 900001 | 0.681 | 0.26 | 0.16 |
iFluor® 680-Wheat Germ Agglutinin (WGA) Conjugate | 684 | 701 | 2200001 | 0.231 | 0.097 | 0.094 |
iFluor® 700-Wheat Germ Agglutinin (WGA) Conjugate | 690 | 713 | 2200001 | 0.231 | 0.09 | 0.04 |
iFluor® 750-Wheat Germ Agglutinin (WGA) Conjugate | 757 | 779 | 2750001 | 0.121 | 0.044 | 0.039 |
iFluor® 790-Wheat Germ Agglutinin (WGA) Conjugate | 787 | 812 | 2500001 | 0.131 | 0.1 | 0.09 |
Citations
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
Authors: Ermatinger, Sarah
Journal: (2024)
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
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
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
Authors: Wijetunge, Sashini S and Wen, Jianchuan and Yeh, Chih-Ko and Sun, Yuyu
Journal: Colloids and surfaces. B, Biointerfaces (2020): 110572
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)
Authors: Killilea, David W and McQueen, Rebecca and Abegania, Judi R
Journal: Journal of food science (2020): 808-815
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
Authors: Guevara, Rebekah B and Fox, Barbara A and Bzik, David J
Journal: mSphere (2020)