Hello Madhuri Kanavalli

You may use RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor. To the isolated mitochondria you may add 200ul of 1X RIPA buffer placed in 1.5ml tube and homogenize with a pestle.

You may prepare RIPA buffer (1X) as follows.

50mM Tris-HCL, pH 7.6

150mM NaCl

1% NP-40 or Triton X-100

0.5% Sodium deoxycholate

0.1% SDS

1mM EDTA

Add protease inhibitor cocktail and phosphatase inhibitor fresh to the ice-cold RIPA buffer when you about to carry out mitochondrial lysis under cold condition.

You may add protease inhibitors such as Pepstatin A (1uM), Leupeptin (10uM) and PMSF (0.1mM) in place of protease inhibitor cocktail. The phosphatase inhibitors to be added include Sodium fluoride (10 mM) and Sodium orthovanadate (1mM).

Best.

Malcolm Nobre Hi... Sorry to piggybank on this question but I was wondering if you could help me out with a couple of questions regarding isolation of mitochondria . The kit I chose to use for isolation (Thermofisher ) doesn't provide any storage buffer to store the mitochondrial pellet . Do you know how I can store the pellets or of a storage buffer ? Thank you for your time.

Madhuri Kanavalli There are several methods you can use to lyse HEK293 cells without sonication. Here are a few options:

Whichever method you choose, it is important to keep the cells and lysis buffer on ice and to work quickly to minimize the denaturation of your protein. You may also want to consider using a protein stabilizing agent such as protease inhibitors to help prevent protein degradation.

Nimisha Pradeep George Isolating mitochondria can be a useful technique for studying the function and biochemistry of these organelles.

Once you have isolated the mitochondria, you will need to store them properly in order to maintain their integrity and prevent degradation. Here are a few tips for storing isolated mitochondria:

I hope this information is helpful. Let me know if you have any other questions.

Ahmad Al Khraisat, Thanks a lot for the detailed answer. I managed to extract my protein using detergent lysis. I appreciate the detailed explanation and tips about mitochondrial storage and buffer conditions.

Ahmad Al Khraisat, I am just curious, is it a good idea to freeze mitochondria as you suggested and extract protein a week later?

Madhuri Kanavalli you mentioned for this point Store the mitochondria: You can store the aliquoted mitochondria at 4°C for short-term storage (1-2 days) or in a -80°C freezer for longer-term storage (up to several months). Be sure to label the samples with the date and any other relevant information.

Yes, that is a common method for storing mitochondria for short or long-term use. It is important to use aliquoted samples, meaning that the mitochondria are divided into small, fixed-volume samples, and stored at a low temperature (4°C or -80°C) to prevent degradation or damage. Labeling the samples with the date and other relevant information, such as the type of sample, is also important for maintaining sample integrity and for tracking the samples over time. It is also a good practice to thaw the samples slowly and avoid multiple freeze-thaw cycles.