I tried to isolate mitochondria from yeast, following a protocol available in jove.com, by Christopher Gregg. However I only had access to lyticase, not zymolyase (which seems to be better in action). After 1h30 incubation with lyticase I tried a teflon first and, on a 2nd attempt, a glass pestle to break the cells but wasn’t successful. Not even a desperate attempt to vortex them worked. Glass beads will destroy mitochondria according to what I’ve read. If you have any suggestion I’d appreciate it very much. Thank you
Hello Ana,
zymolyase is indeed more efficient for yeast lysis than lyticase. But it is possible to use lyticase for cell lysis as well. You should then use a relatively high lyticase concentration (e. g. 300 U/ml) for at least 70 min at 30 °C with gentle shaking (~100 rpm).
Best regards!
Hi! thanks for your answer! I forgot to write above that I used 3x more lyticase. So, in the protocol they use zym 100U/g pellet and I used lyt 300U/g for 1h30, at 30C and 80rpm. These S. cerevisiae are tough... :S
This link might help you:
http://www.amsbio.com/brochures/Zymolyase_Comparison.pdf
Also, try to use fresh cultures. The cell walls in old cultures tend to be thicker.
Hi! I have seen this document before and I thought it was quite helpful. It was because of it that I decided to use more lyticase. :)
My goal is to study the cells in stationary phase, but log phase will be a possibility if I run out of options. :\
Thank you for your suggestions.
Hi Ana,
Sometimes the same happened to me even with Zymoliase. For that reason i'm now doing the incubation of cells with Zymoliase at 32ºC with DTT or Mercaptoethanol for 30-45 min. I also check the cell wall digestion with SDS.
Hi!
Till: Yes, in this case DTT buffer (10mM) is added before, for 20min, 70rpm, 30C. Then it is fully removed. Increasing the lyticase concentration is a good option but lyticase is a bit expensive so I was trying to avoid doing so. I will try the water trick. :)
António: Thank you for sharing that zymolyase is not working all the time. I was considering to buy it (...not sure I could, anyways). I thought that it might be my main problem. I had no idea I could do the incubation with lyticase and DTT at the same time. What concentration of DTT do you use in this case?
Thank you guys! This thread is being quite helpful. :)
In my lab we suspend the cell pellet in the suspension buffer (containing Sorbitol, Sodium phosphate and EDTA) and we add Zymoliase and 1 µl of ß-mercaptoethanol per 100 µl yeast suspension. In the case of DTT the final concentration that we use is 20mM. Then the suspension is incubated at 32ºC until the majority of cells is digested.
A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation
Yuchong Wang, Kathryn S. Lilley and Stephen G. Oliver
DOI: 10.1002/yea.3002
Yeast, Volume 31, Issue 4, pages 127–135, April 2014
Hello!
Till: the lyticase buffer contains 1.2M sorbitol. Why do you ask? Will it interfere a lot with the OD readings?
António: I will try the DTT solution. Do you think it will make a big difference using 10 or 20mM? I wonder because my DTT buffer for pre treatment has 10mM only. Regarding EDTA, I add to the homogenization buffer (next step), but not to the lyticase buffer (sorbitol and PBS only).
Elizabeth: Interesting procedure in the paper, though I have no access to this cell disruption vessel, nor I know how to do it :S Have you ever done the procedure in this lab? I actually thought of going to another to do the isolation but then it would be hard to bring the mitochondria back to my lab...
Thank you all!
Hi Ana,
I don't think that it would be very different either you use 10 or 20 mM of DTT, but try also to test Mercaptoethanol. Normally I have better results.
You can also take in account another aspect of Lyticase. Its pH optimum is 7.5 and its activity drops off rapidly below that, decreasing the spheroplast formation due to cellular metabolites that acidify the buffer. Maybe you can check and adjust the pH during the incubation step to increase the efficiency of lyticase.
Update: this afternoon I started the experiment again and I now think it is the pH. From the beginning the media was around pH 6. I added NaOH (what I had available at the moment, instead of KOH), and added more after 1h, as the pH dropped again. Finally I could see the separation of "phases" with the translucent part on the top. I realized today that zymolyase has an optimum activity at around pH 5.8, so it would have probably worked if I had had the opportunity to use it. oh, bummer...
After a few strokes with the pestle and centrifugation I still have a small mitochondria pellet, but well... that is the next challenge...
How about freezing your material before you grind it? It might help break the cells .
well, I tried that too, freezing after growth, though mitochondria don't like it... And got the same results.
Hi Ana,
When you use the lyticase, it is freshly prepared or was frozen? Frozen lyticase loss quickly the activity.
Good morning,
I use it frozen. Never thought of such problem because we use lyticase for other protocols and it has always worked fine ...though I would have to do some 'calculations' in order to figure how much U are usually used. (When in small quantities, one abuses of volumes without thinking of costs. :S)
Hi again Ana,
Lyticase is not so good as Zymoliase but you can use it if you prepare freshly the solution of enzyme. When you buy (from Sigma probably) is a lyophilized powder that can be store a -20ºC several months. When you use the enzyme prepare the amount that you need and the rest it is possible store in solution at 4ºC several weeks.
However, the best is change to Zymoliase 20T that is better that Lyticase
See you
Good luck
please find attached the method we use in our lab. Should you have any questions do not hestitate to contact me.
- Badges
- Science topic