For a protocol, I need to proceed to the lysis of a lysis of a sample of mouse mitochondria. They are retrieved from mouse liver by using QIAgen Qproteome mitochondria purification kit and finally resustended in PBS.
So far i tried 3 different approaches:
- By osmosis, using distilled water,
- with a mitochondrial lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% (v/v) Triton X-100, 0.3% NP-40, PMSF, protease inhibitor cocktail)
- By sonication following Zhang et al. (Nature, 2010) protocol: 10x 30sec. at maximal amplitude with 30s intervals)
I checked for remaining mitochondria with our cytometer (CantoII special order, upgraded for small particles) and none of these methods gave satisfying results, so my question is:
Do someone knows/uses an efficient protocol? I'd be especially interested by one that doesn't use detergents.
Thanks a lot!
Thanks for the advice and would you have an idea for a buffer that doesn't include detergents?
We are normally using SDS loading buffer that is used for normal SDS-PAGE gels and it works well. If you are using RIPA which has detergents, you would consider doing TCA too.
SDS and Sod deoxycholate in RIPA buffer are stronger detergents and should work. The higher the concentration you use the stronger the lysis, but they tend to interfere with protein assay if you plan to do it later. Sonication can be very strong depending on the power you use. You can combine it with osmosis by doing it in distilled water.
Yes, that is my problem, among the experiments I would like to run are ELISA assays where some detergents may interfere (hence the reason of my question about lysis buffer with and without detergents)
Thanks everyone for your replies!
May I suggest the olden days of multiple (may be two or more cycles) freeze thawaing as a non-interferring method for proper lysis?
2% CHAPS in Tris buffer works well. The mitochondria isolation kit from ThermoFisher recommend it to lyse mitochondria and quantity protein.
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