I have studied about the mitochondrial enzyme in yeast. So, I want to extract the crude enzyme from cell culture in order to test the enzyme activity. What is the suitable procedure in this case? Thank you very much.
So do you want total (crude extract) or mitochondrial proteins?
If you know your protein is in the mitochondrion, it would be better to work only with mitochondria.
In this paper they describe the isolation of mitochondria from yeast (Pallotta 2011)
http://onlinelibrary.wiley.com/doi/10.1002/yea.1897/abstract
If you want crude extract, there are several options:
- enzymatic degradation of cell wall
- chemical degradation of cell wall
- disruption by glass beads on vortex
- ultrasound
I'm not sure, whether freezing and thawing works for yeast, but since it works for bacteria, it probably should.
You'll have to check which one works for your protein and which one is suitable for you (depending on how much and how often will you perform this).
Hi there,
For a quick crude lysate preparation, glass bead treatment is the easiest.
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