Wheat Germ Agglutinin as a Plasma Membrane Marker

December 11, 2015

Colorado State University
Food Science and Human Nutrition
Graduate Student

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Company:

Thermo Fisher Scientific

Product Name:

Wheat Germ Agglutinin, Alexa Fluor 350 Conjugate

Catalog Number:

W11263

I am comparing the size of the endoplasmic reticulum to the size of the whole cell in a variety of treatment conditions. The ER was successfully labeled using a GFP transfection probe but having a plasma membrane marker to accurately compare the size of the organelle to the whole cell is crucial. This product was used as a potential blue plasma membrane stain.

Experimental Design and Results Summary

Application

Fluorescent Microscopy

Starting Material

H4IIE rat hepatoma cells fixed to a coverslip

Protocol Overview

After cells were fixed to a coverslip, the wheat germ agglutinin (WGA) is diluted (recommended dilution is 5ug/mL) and allowed to incubate on the fixed cells for a certain period of time (recommended is 10 minutes). Then remove the WGA and rinse 3 times with buffer of choice (recommended is HBSS) and mount coverslip onto slide.

Tips

None

Results Summary

After following their recommended protocol exactly, the conjugate was very faint. I thought it didn't even work at first. I had to bump up the exposure time on the microscope to 15 seconds before I could see it. I then repeated the experiment and increased the concentration as well as the time the WGA was exposed to the cells. This made little difference. I was able to view the cells with about a 5 second exposure time. This product may work for some but I need to take a photo of both the ER and whole cell and this long of an exposure photo bleaches the cells too much.

Additional Notes

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Summary

The Good

I have heard a lot of good things about this product as it seems to work to target the plasma membrane in a variety of cell types.

The Bad

In this case, the WGA is too faint and not readily detectable so I had issues with photo bleaching.

The Bottom Line

WGA does not bleed into organelles within the cell because it is a protein and hydrophobic, unlike other plasma membrane stains. However, it was hard to distinguish between cells (confluency was 80%) and photobleaching was a large issue. Maybe would work better for others.

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