Choco Michael Pagayatan Gorospe

Umeå University | UMU · Department of Medical Biochemistry and Biophysics

I was wondering if anyone has tried to G2 synchronization of yeast cells using the drug RO-3306. And if the drug works, as in mammalian cells, what concentrations have you used and treatment time.

Thank you.

I would like to measure mitochondrial mass, and wanted to use the Nonyl Acridine Orange (Acridine Orange 10-Nonyl Bromide).

I have used the MitoTracker Green for measurement, although it might be independent to membrane potential, it seems to be interacting with the uncouplers like FCCP and BAM15. As addition of the uncouplers reduce MTG values in flow cytometry.

I would like to ask if someone has observed the same using the

Nonyl Acridine Orange (Acridine Orange 10-Nonyl Bromide)?

Hi everyone, I hope everyone can help me with this.

First, I am measuring mitochondrial membrane potential by flow cytometry using TMRE and MTG dyes. I think we are not experiencing problems with my TMRE readings. However, I have problems with my MTG readings. Supposed to be that MTG is independent with the membrane potential. But, if I add uncouplers (CCCP), the values for MTG stained cells are significant lower compared to CCCP untreated samples (30-50% lower).

My procedure is, I stain the cells first with MTG and add the uncoupler (30uM) after 20 mins, and then incubate it for another 10mins (total of 30mins for the dye).

There are 2 possible scenarios that I am thinking.

First, the dye and CCCP interact with each other leading to a lower fluorescence intensity readouts.

or,

Second, the uncoupler cause severe mitochondrial damage that it initiates mitophagy.

but is 10 mins exposure to the uncoupler (plus few more minutes while waiting it to be read in flow cytometer) enough time already to be degraded by mitophagy? or is the first scenario a more logical explanation for the lower values in CCCP treated cells?

Hi colleagues,

I am trying to measure mitochondrial membrane potential and mitochondrial mass in S. cerevisiae with TMRE and mitotrackerGreen as dyes using flow cytometry. However, I always see two peaks when measuring them.

I have been trying to optimize my protocol to prevent or reduce the double peaks but still not successful.

Has anyone observed these same results in their experiments in yeasts? And how did you deal with it?

So far, what I have seen in other publications are single peaks, and I am not sure if they gated out the other peak and if I have to gate it out, which of those peaks have to be gated out?

I was also thinking it is probably because of doublets, or maybe unbudded cells, or aggregated cells. And I have tried to address them by sonication, adding Tween20 and EDTA, but it did not resolve the problem.

I am checking expression of autophagy -related proteins in yeast. To do so, I cultured and harvested cells in early stationary phase and very late stage (4 day culture). I planned to do western blot but I could not detect proteins on the short-lived strains I harvested at day 4 of culture using bradford assay. I prepared another lysate from a different sample of the same strain but detection is either 0 or close to zero in bradford. so I was wondering if the cells die (either apoptotic or necrotic), proteins can not be detected anymore. I am not actually sure if they actually died (just hypothetic since they are chronologically short-lived cells) though I senescent cells are also of possibility.

I have been doing experiments related to expressions of TOR1 in yeasts. in our lab, we were able to detect to TOR1 using TOR1 antibody but suddenly we had difficulty detecting it. We thought the problem is the antibody because we ruled out every other things that could become the problem. We have no other options since only one company is selling it (we don't know any other company). We bought a new one but also not detecting the protein.

 I tried to TAP-tagged the TOR1 in C-terminal. It was successful and able to detect the tagged protein. but the problem is, we are observing weird phenotypes that are not related to its normal strain. It could be that the tagging made some undesirable effects on the protein activity.

I would like to ask what other labs are doing in dealing with TOR1 expression or what you did just to detect the protein.

Thanks in advance.

I usually do my blocking for 1 hour at RT using 5% skim milk, but now I am doing it already for 2 hours or so because the background is not clean with just an hour blocking. My skim milk powder is somewhat old, we bought it last 2011. Is it possible to increase the concentration of the powder or should I just buy a new one.

I am making a tag for ATG8 for my western blot analysis. I was able to construct ATG7 and ATG1 tagged with TAP but not with ATG8. I was checking on some papers and noticed that all or almost all of ATG8 was tagged with GFP but not TAP. I know that GFP can be used for fluorescence studies but some GFP-ATG8 papers I searched did not have fluorescence studies, only western blot.