What is the suitable temperature for keeping yeast liquid culture after incubating at 30c, 250 rpm for overnight? The type of yeast is saccharomyces cerevisiae. I plan to keep it first for a few days before starting to extract candidate protein from yeast liquid culture and mix it with sample buffer prior to perform sds page for determination of candidate protein size. Thank you.
Keep the culture in 4C refrigerator. I think it is better to harvest the strain and keep in -20C
can I keep yeast liquid culture at -80c? after a few days, I will take out the tubes containing yeast liquid culture from -80c freezer before protein extraction.
You can store in -20 deg but just the cell free broth. Try adding some kind of protease inhibitor or adjusting pH of broth before storage .
Instead of keeping the complete liquid culture, better you harvest the cells from culture and you can store the cell pellet in -80c for a long time and let it to thaw in ice before going for protein extraction.
Good Luck..!!!!
Usually, I keep the harvested strain in -20 or -80 for a few days, not the liquid culture. So, I donot know whether that will be OK.
You can keep the liquid culture in 4 degree for few days..or can pellet down the cells and keep the pellet in -20 degree for long tern storage...or you can also re suspend you pellet in compatible buffer the store it -20 or -80 for long term storage..
If you are keeping your cell pellet in -20 or -80 make sure you add glycerol to the pellet and mix it otherwise cells ll damage due to ice microcrystals.
Dear Anshul,
Addition of glycerol is not required for yeast cells because it's cell wall is very tough/harder.
For maintaining the strains for future use, it's better to make 50% glycerol stock of that strain. But, glycerol is not required for cells that you use for protein extract preparations.
I didn't actually took in consideration about the source of protein extraction ( extracellular or intracellular) ... but yes ,you are right ,glycerol is surely added for the stock storage at -80.
You can store your liquid culture for a couple of days in the fridge (4ºC), but I think it is best to harvest the cells and store the pellet at -20ºC.
One important thing to keep in mind, in my opinion, is the kind of protein against which you want to test. For example, I could imagine that fresh cells are better than a frozen pellet when you want to test for any stress-related protein. In other words, can you be sure that your cooling/freezing has no impact on cellular protein levels that you are interested in? There are also yeast mutants that are cold-sensitive and start dying on plates in the fridge, which means that the number of healthy cells in your pellet decreases and the amount of degraded material will increase.
There are also many protocols for rapid yeast lysis and protein extraction, so you might consider processing your cells immediately and store the extracts.
Maria makes a good point, the most important thing for you to consider is: at what growth stage is the protein you want to isolate expressed or maximally expressed.
This is the factor that will determine your procedures more than anything else.
Generally if I am generating cells to purify protein from I will grow a large overnight culture (the protein we isolate is highly expressed so we don't care too much about growth stage at harvest), harvest by centrifugation, wash twice with water and freeze at -80 in either water or lysis buffer depending on how we are going to lyse the cells.
You only need to add glycerol to cells that you freeze if you want them to live once thawed. However if you want to freeze a protein extract from the cells you should add 10% glycerol to your buffer prior to freezing.
Other than that, we routinely keep liquid cultures and plates with colonies on them for up to 4 weeks at 4 degrees.
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