Hi everyone, I am hoping to take a look at the cell cycle progression for my budding yeast and I have chosen alpha factor as the method of cell cycle synchronization. I wish to ask for your help because one of the mutant I am currently working with simply won't arrest in response to alpha factor.
The issue: mutant unresponsive to alpha factor, after 150mins arrest, only 50% of the total population are shmoos (~80% unbudded); when I took a look at the cell culture with flow cytometry (PI stained), a lot of cells seems to be stuck between G1 and G2. When the same alpha factor concentration is applied to WT, it arrest as expected.
Experimental condition: I used buffered YPD media (pH~5). One the day of the experiment, I diluted my cell culture (~1.5 OD600) back down to 0.1OD, allow it to double once before adding the alpha factor for arrest.
Optimization I have attempted: I have played around with the alpha factor concentration and arrest time but strangely none of them seems to affect the arrest efficiency. I still get one peak between G1 and G2 for my mutants.
Therefore, I wish to check in with everyone on research gate to see if someone can give me some pointers for how to further increase my alpha factor arrest efficiency.
Please let me know if there's any clarification or additional information I can provide. Thank you in advance!
Hi there,
Your strain seems to be deficient for cell arrest induced by alpha factor (deficient for some STE proteins of the response pathway?). What is its genotype? You might try to introduce bar1 deletion in your strain in order to improve cell sensitivity to alpha factor. Otherwise you may try other block and release strategies (hydroxyurea in S phase or nocodazole at G2/M).
Dominique Liger Thanks for the suggestion and for the help! my strain is gcn5Δ, I will definitely look into if this mutation causes any arrest deficiency. Is there any arrest strategy/ protocol you would recommend? for now I am considering Nocodazole arrest at G2/M. Thanks again for all your help Dominique
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