Hello everyone,
For my research I’m culturing renal epithelial cells and wanted to observe the primary cilia formed by the microtubules, however a curtain subpopulation does not stain positive (or greatly reduced expression) for alpha-Tubulin.
Does anybody know what could be going on?
I’ve added a picture of the staining.
I was thinking that the epitope could be post-modified and the antibody no longer binds.
The antibody I’m employing is the ab18251 from Abcam, I still have to receive answer of Abcam if post-modification could explain the negative population.
However is it then possible that all microtubules are post-modified and no longer visible? Or is something else going on?
Best,
Ronald
Ronald C. Van Gaal
Hi. Cell cultures are mostly not a homogeneous cell population. Populations in cell cultures can be terminally differentiated. Or populations can be able to grow and divide, which is not generally associated with mature epithelial cells.
However, most kidney epithelial cells in vivo are in tight columnar formations and it could be that your cells are not confluent enough for all of them to coordinate expression.
Hi Ronald C. Van Gaal ,
Very interesting observations indeed. I just had a quick look at the tubulin antibody product sheet you are using and think that there is a good chance of the antibody binding being affected by tubulin PTMs. Alternatively, it cannot be ignored that the cells might be expressing different tubulin isotypes (there exists different tubulin isotypes, mostly divergent in the c-terminal part of the protein).
One way to understand this is to simply use tubulin antibody from another source. At the same time, you could also try to co-stain these cultures with tubulin PTM antibodies. Please see this reference for more details
"The tubulin code: Molecular components, readout mechanisms, and functions"
However, before concluding something, it will be better to co-stain these cultures with actin or other cellular markers (preferably apoptosis markers) and make sure that the cells showing negative staining for tubulin are alive and healthy.
Thanks
Satish
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