What is a good amount of tissue to start with?
I am looking for a protocol for isolation of pure mitochondrial fraction from mouse kidneys. I was also wondering whether I need to start with one or 2 kidneys to get a good yield. Thanks!
We used the protocol for the buffers of A.M. Silva and P.J. Oliveira published in Mitochondrial Bioenergetics (in general a great book for mitochondrial work) and had nice results for both rat and pig tissue. Otherwise you might want to have a look at: A semi-automated method for isolating functionally intact mitochondria
from cultured cells and tissue biopsies.
I can sent you the extended version of our protocol if you want to give it a try. The amount of tissue you need depends on what you did with the animals before and what you are planning to do with the mitochondria. Our extended protocol is for respiration measurements.
Thank you both.
I am testing whether my protein of interest is in mitochondria or not, so I need a protocol that should give me a pure fraction. I noticed that the paper from Pamela do not involve gradient centrifugation. How pure are your mitochondria?
Maximilia, do you know if you protocol gives a pure fraction? How many kidneys do you suggest to use to look for mitochondrial protein content?
I have to admit we never tested. Since I don't look at proteins I don't know if one is enough.
I need a higher grade of purity. Maybe I can start with 2 kidneys and Percoll?
We use dounce homogenization, differential centrifugation and afterwards a two-step sucrose gradient ultracentrifugation - works fine on one mouse kidney and gives mitochondria with no nuclear contamination detectable by our Western blots. So should be working fine for your purpose hopefully. Tell if you need a detailed protocol
Thanks Steffi. I would like to try your protocol if this is fine with you. Can I find it in a publication or can you send it to me?
We have the protocols for mouse liver. This protocol had also been proved to be useful for isolating the mouse skeletal muscle as well as the flight muscles of migratory locusts, so, in my opinion, it will also be valuable for mitochondria isolation form mouse kidney.
This protocol is described as followed:
• Isolating buffer: pH 7.4
Sucrose 250 mM
Hepes 10 mM
EDTA 0.5 mM
BSA (fatty acid free) 0.1%
• Procedure:
1. Starve the mouse overnight before the isolation experiment.
2. Kill an adult mouse (about 3-4 months) by cervical dislocation and rapidly explant the liver from the peritoneal cavity. Find the gallbladder and remove it using a scalpel. Immerse the liver in 50 ml of ice-cold Isolation Buffer (IB) in a small beaker.
3. Rinse the liver free of blood by washing 4-5 times with ice-cold IB.
4. Mince the liver into small pieces using scissors.
5. Discard the IB used during the mincing and replace it with 5 ml of ice-cold fresh IB. Transfer the suspension to the glass potter.
6. Homogenize the liver using a Teflon pestle operated at 2000 rpm., stroke the minced liver 3 times.
7. Transfer the homogenate to a 50 ml polypropylene Falcon tube and centrifuge at 3000 rpm for 10 min at 4 °C.
8. Transfer the supernatant to a new 50 ml tube and centrifuge at 10000rpm for 10 min at 4 °C.
9. Discard the supernatant. Then resuspend the resident of mitochondria in 10ml ice-cold fresh IB, and centrifuge at 10000rpm for 10 min at 4 °C again.
10. Discard the supernatant, and resuspend the resident in 1 ml ice-cold fresh IB.
• Purification of Mitochondria by Percoll Gradient Centrifuge
1. Prepare a linear Percoll gradient of 8-60% (V/V) buffered with 0.25 mM Sucrose, 0.2% BSA (fatty acid free) and 10 mM Tris-HCl, pH 7.5 using a gradient maker
2. Gently but thoroughly re-suspend the crude mitochoindria pellet in 0.8 M Buffered sucrose and lay on the top.
3. Centrifuge the gradients at 37,000 g for 20 min at 4℃.
4. Remove the mitochondria band and dilute it with 2 volume of the gradient buffer and pellet the mitochondria by centrifuge at 25,000 g for 10 min at 4℃ (or 6300g for sever times)
5. Wash with isotonic buffer to remove Percoll
6. Resuspend the purified mitochondria in MSB buffer containing 400 mM mannitol, 10 mM KH2PO4，50 mM Tris-HCl，pH 7.2 and 5 mg/ml BSA (fatty acid free).
Dear colleagues,
I have the same problem. We investigate respiration of mice mitochondria from different tissues. In some articles ("Isolation of Liver or Kidney Mitochondria", Johnson&Lardy or "Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse" ) only cortical mitochondria are used. And medulla is thrown away. Why? Is it possible to use the whole kidney?
Thanks in advance!
Hi, we're isolating mitos from mouse kidney regularly using the whole kidney. One kidney is good enough for any of our purposes, and the isolation is fairly easy.
We do the commonly used protocol: chopping with scissors, isotonic homogenization with a Dounce homogenizer, differential centrifugation and a two-step sucrose centrifugation. We haven't checked any respiration, but proteins and mtDNA are intact and pure afterwards.
Dear Steffi, thank you for your answer! We tried this protocol for isolation. It seems to be good. But there is an idea that cortical homogenate is used, because there are more mitochondria compared to medulla. And this can have sense during oximetry. But still I'm not sure..
Hi, I am now using a protocol for isolation of both mitochondria and MAMs and it works pretty well using whole mouse kidney (Wieckowski at al., 2009 Nat Protocols). Not sure why you would need to use only cortical, is it possible they were looking at specific nephron segment?
here's our standard protocol that in our hands works very well for mosue kindey. Usually I take both kidneys from one animal, but one is enough if you don't need masses of mitos for your measurement
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