Currently, I am working on yeast strain CEN PK and I want to carry out TUNEL Assay to detect amount of damage upon treatment with some damaging agent. We have a kit in the name of TUNEL Assay Kit - In situ Direct DNA Fragmentation (ab66108) and the protocol in this kit is specific for mammalian cells.
I tried adding a step after fixation that includes Zymolyase treatment in order to remove cell wall and expected to get results. (Reason being yeast cell wall might hinder permeabilization of kit chemicals). But I am not successful in getting results.
So, can someone provide a working protocol for yeast in order to perform TUNEL Assay?
or provide suggestions.
Thank you.
Hello Jyoti.
Critical steps are complete permeabilization, HCl treatment and complete removal of trace amount of HCl at subsequent washing procedure. If the once entrapped acidic condition is not released, antibody would not get in under acidic condition.
See whether you can achieve TUNEL in mammalian cells, but not in yeast by proceeeding two samples together. The enzyme should be included in the yeast. Then you will get the answer.
Regards.
Dear Jyoti.
you can follow this link:
Ribeiro GF, Côrte-Real M, Johansson B. Characterization of DNA damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock. Molecular biology of the cell. 2006 Oct 1;17(10):4584-91.
Best
Hello Jyoti,
I agree with Zahra. The paper by Ribero et.al. gives a comprehensive protocol to perform TUNEL staining on yeast. The authors use zymolase to permeablize yeast cell walls following fixing the cells, solves the major hurdle of getting the reagents in the cells for staining.
Best of Luck!
Ashutosh
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