I am following the standard method which is mentioned in Tomotake Kanki et al., 2009 research paper Article Monitoring mitophagy in yeast: The Om45-GFP processing assay
. I am growing the cells in SD-HIS selective media, then transferred into YPD, then pelleted, washed (2xPBS), Then again transferred to YPL. After keeping cells in YPL for 12-14 hours (To reach mid log phase), again pelleted, washed (2xPBS), transferred to SD-N for 6 hours.I have tried this method so far, Even some times, changed the methodology like grown the cells in selective media such as SD-HIS media, from there, cells were pelleted, washed two time with PBS, then keeping the cells in YPL for 12-14 hours to reach mid log phase, again pelleted, washed (2xPBS), transferred to SD-N for 6 - 8 hours. But here also, no favorable results.
However, In every literature related to mitophagy inducing they have mentioned a YPL media ingredient as YPL; 1% yeast extract, 2% peptone, 2% lactate. Other than that Tomotake Kanki et al., 2009 Article Monitoring mitophagy in yeast: The Om45-GFP processing assay
" Monitoring mitophagy in yeast The Om45-GFP processing assay" mentioned as YPL; 1% yeast extract, 2% peptone, 2% lactic acid, pH 5.5. I believe that, in other literature they meant lactate as lactic acid, or do we really have to use lactate such as sodium lactate? While preparing for the YPL media, I carefully adjusted the pH at 5.5 to 6.In above, I noticed that, cells in YPL media do not grow like YPD, even though it seems like there is no significant growth in YPL media, then how in literature is it mentioned as growth till mid log phase in YPL?
After inducing mitophagy in SD-N, when I look at microscopy (100X), I am seeing the cells which it looks like control (without Nitrogen starvation). What do I have to do?
At least, in the western blot also, I am not getting free GFP at 27 KDa. I am using 0.45 micron nitrocellulose membrane. Can anyone please help me with my doubts? I am trying hard to get the results for mitophagy induction.
Attached files contains, OM45 2 g is under blue light filter, om45 r under green light filter (FM64-4, Vacuole dye), om45 2 b is bright field. Even when I use FM64-4 to track vacuole, under blue light also, I can see that red fluorescence in the vacuole, I am using 16 micromolar for 1 * 10 power 7 cells/ml.
Another important thing which I noticed that, while autoclaving SD-HIS media, it is forming sedimentation after autoclaving, it leads to artifacts while seeing under microscopy, those artifacts exposing light so much.
Please anyone help me with this. I am very grateful to you.
Thanks in advance.
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