Hello I am currently trying to set up a new protocol. Our lab is planning to perform study about mitochondria, and we still couldn't set up a proper protocol.
The kit we used is from abcam (ab110168), and since the protocol is written based on large amount of tissue, we downsized the protocol to 1/10 amount of tissue. (about 30mg)
1. Wash and mince the tissue, and then homogenize the tissue using dounce homogenizer. Before homogenizing we put isolation buffer until the total amount is 200ul.
2. Put the homogenate in an e-tube, then add isolation buffer until the total amount is 200ul. Then centrifuge for 10min, 1000g, 4℃.
3. Add isolation buffer to the supernatant until the total volume reaches 200ul. Then centrifuge for 15min, 12000g, 4℃.
4. Collect the supernatant for it is the cytosolic fraction, and add isolation buffer 100ul+protease inhibitor cocktail 1ul to the pellet. Then centrifuge for 15min, 12000g, 4℃.
5. Discard the supernatant, then add isolation buffer 100ul+protease inhibitor cocktail 1ul. Then centrifuge for 15min, 12000g, 4℃.
6. Discard the supernatant, add isolation buffer 49.5ul+protease inhibitor cocktail 0.5ul and resuspend the mitochondrial pellet.
While homogenizing, we stroked the pestle for total of 40-50 times. After that we tried decreasing the number of stroke to total of 10 times. Either way, when we performed western blot to check the purity, we could see the band of cytochrome c from cytosolic fraction, and GAPDH or b-actin from mitochondrial fraction.
After that we used another kit from abcam(ab65321) which is for mitochondrial DNA isolation. the protocol was slightly different and we stroked the pestle for total of 100 times. We used the same amount of tissue (about 30mg) and we could still see b-actin from mitochondrial fraction after western blotting. I should mention that the reagents we used from this kit was opened in 2019. Also the speed for high speed centrifuge was 10000g, and the time was 30min.
Is there something wrong with our protocol? Or is there anyone who successfully isolated mitochondria from the cytosolic fraction?
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