Hi! You can check out this Nature Protocols article, which has a detailed description for a procedure of getting these from both cells and tissues (liver).

http://www.nature.com/nprot/journal/v4/n11/full/nprot.2009.151.html

HI!

You can check our paper which has a description for mitochondria:

Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal musculus.

To open the mitochondrial outer membrane, from isolated mitochondria you should use 20 mM HEPES-KOH pH 7.2 with 20 minutes of incubation on ice.

The mitoplasts (pellet), were isolated by centrifugation for 30 minutes at 64,000 g (2°C). In the solution you will have fraction of outer membrane. If you want also recover these, frozen them in liquid nitrogen and perform lyophilization

We have not checked in the mouse but we have seen this with rat liver mitochondria, in a phenomenon known as high amplitude swelling, HAS. Dilutions of mitochondria way below 1mg/ml result in large changes in turbidity called HAS, which actually was shown by us to be serial disruption of mitochondrial membranes, driven by colloidal swelling induced by mitochondrial respiration and the low ionic strength of sucrose media leading to electrostatic repulsion. First the outer membranes rupture, followed by loss of intermitochodrial proteins and cristal unfolding (maximal turbidity change) and lastly by breaking of the inner membrane. Density gradient centrifugation indicates different fragments and the existing fragments show anomalous interactions between mitochondrial proteins. The advantage however is that you don't add anything and whatever you get is free of external contaminants.

Hi Marco!

I have inserted the biblographic source to a reference below that may be of use for you. This is a in vitro method, however, I am positive that you will be able to adapt it to your mouse liver tissue. Good luck!

Reference: Ahn Y-H, Koh J-Y and Hong SH. Protein synthesis-dependent but Bcl-2-independent cytochrome c release in zinc depletion-induced neuronal apoptosis.

J Neurosci Res. 2000;61:508-51.

The most reliable way of separating functional outer and inner membranes in my experience is to break isolated mitochondria in a French press at 2000 psi. (735 at medium hight). Then you will need to lay the broken mitos on a 35 % sucrose cushion (in 150mM KCl or isolation buffer, depending on your needs) and spin at 25000 rpm for 1h. The top layer will be outer membranes and the inner membranes will be in the pellet. Clearing centrifugation steps at the same speed for 30 min will remove sucrose.

Best of luck,

Pablo.

Hello Marco!

As people have already advised, the best way to blow mitochondria and release inter membrane and intramitochondrial proteins is to cause large amplitude swelling. The incubation medium might contain either 20 mM MOPS, pH 7.2, or 10 mM MOPS + 10 mM sucrose or KCl. MOPS, to my opinion, is bnetter than HEPES because at pH 7.2 it has maximum buffer capacity. Whether to use sucrose or KCl you have to play, in order to achieve maximal detachment of proteins from the membranes. Use porin as a marker for outer membrane and determine the relative amount for different fractions. These fractions are obtained at different accelerations. The largest g is about that for the microsomal fraction. To avoid unnecessary contamination, use only middle fraction of the liver mitochondria, that is between 2000 g and 8-9000 g. The lighter mitochondria will contain lysosomes and peroxisomes in addition to the light fraction of mitochondria.

You can use also a marker for the inner mitochondria as a contaminant. For this would be good to use any antibody against cytochrome oxidase .

Panov's response is okay with two exceptions. If I remember correctly, beef heart mito do not show equivalent swelling. So you need to check that with the mouse mito. Secondly, HAS is an electrostatistically driven phenomenon in respiring mito. So you cannot use electrolytes of any kind since about 10-15 mM NaCl equivalents will inhibit the phenomenon. See...

*Respiration induces variable porosity to polyols in the mitochondrial inner membrane. D. Sambasivarao and V.Sitaramam, Biochem.Biophys.Acta. 806, 195-209, 1985.

*Dynamics of instability of mitochondrial membranes during metabolically induced high amplitude swelling. D.Sambasivarao and V. Sitaramam. EBEC Short Reports, 3B, Hannover, ICSU Press. pp. 553-554, 1984.

At the end of the sixties, when I was a PhD student working on mitochondrial anion translocation, there was this by now classical paper of Schaitrman & Greenawalt. Although I never used the method myself I know that many others did, with success. Perhaps you already know this paper. If not, hopefully you will find some useful information in it.

Hi! I know that Dr Roger Castilho has been working with mitoplast. I check for his earlier paper on the subject and found this: J Bioenerg Biomembr. 2011 Dec;43(6):709-15.

Reactive oxygen species and permeability transition pore in rat liver and kidney mitoplasts. Ronchi JA, Vercesi AE, Castilho RF.

Check that out and I am sure you will be able to get the mitoplast.

Best regards

Fernanda

Dear Dr. Vetury Sitaramam,

Dr. Spinazzi wishes to receive outer membrane from mouse LIVER mitochondria.

Otherwise, you are completely right that heart mitochondria and brain mitochondria may not undergo large amplitude swelling at all. Some cancer mitochondria will not swell even in the presence of alamethicin. It seems that only liver mitochondria have large osmotically active matrix space for large amplitude swelling. In addition, I have data that brain mitochondrial swelling is a subject to variation between strains.

Alexander

Dear Dr.Panov,

Yes, I was aware that he was talking about liver.

If I may add, all mitochondria have osmotically active space. Swelling phenomena of an electrostatic kind can be seen in all cells and organelles as well. We have shown that only cardiolopin and cerebrosides confer porosity to PC liposomes. Neuronal membranes swell during action potentials. The phenomenon is more biophysical than biochemical and you should find many of these papers from our lab here.

The method in Fraser & Zammit (Biochem J 329, 225 - 9) works very well for rat liver mitochondria. May need minor adjustments for mouse liver.

Thanks to all for the kind suggestions. I have no problem in releasing isolating intermembrane space proteins after osmotic shock (Hepes 10 mM, pH 7.4) nor in isolating matrix proteins. The problem is specifically in the separation of outer membrane proteins from inner membrane proteins (which do not separate after osmotic schock, which only causes rupture of OMM). I tried extensively the Schaitman & Grenawalt protocol (which was used by Castilho as well) in mouse liver mitochondria, attempted several modifications, different digitinonin stocks, but all failed: OMM were solubilzed at the same time as IMM. I have done extensive reserach in the literature, and I noticed that all the published articles using this technique were on RAT mitochondria, never on MOUSE mitochondria. Therefore, I suspect there might be a species specific limitation. Is anybody aware of it?

Unfortunately the french press in not available in our institute. As an alternative protocol at the moment I could find only the sucrose gradient subfractionation protocol (originally from Sottocasa GL), but the problem is that this protocol is not trivial, and it is not so easy to replicate it. Also, since it was described in the 60's there are not WB of mitochondrial proteins from different subcompartments for quality control. If anybody has hands on experience, or has other suggestions I will be extremely grateful. Best Regards

Try Ardail et al JBC 266, 7978 for mouse mitochondrial membrane fractionation.

Thanks for the suggestion Dr. Zammit, I will try it, although it is a pity that there are not WB for quality control of the different subcompartments. Did your lab test this technique by chance?

We worked entirely on rat mitos, and fro our papers it is obvious that it works very well. I cannot see why it shouldn't work with mouse mitos, although considerable experience is required for it to work, particularly in standardising the (hand) homogenization, the teflon pestle/glass body homogeniser clearances, and the type and duration of sonication. Lots of marker studies need to be performed first, and the resulting optimised protocol adhered to subsequently (and preferably used by the same person too).

Dr. Spinazzi,

Were you able to Isolate mitoplast from OMM? I am trying now on mouse mitochondria isolated from brain but I cannot seem to get rupturing of all mitochondria. I have a lot of VDAC in my mitoplast fraction after performing the sucrose gradient centrifugation. So either I am not rupturing all the mitoplast or the separation of OMM from mitoplast is not good. What are you thoughts?

Hi Simon,

I think that obtaining a clean separation of OMM and IMM is very difficult. Moreover there is a clear terminological ambiguity on the significance of "mitoplast". Many improperly refer to mitochondria with broken outer membranes, while instead this should only refer to mitochondria devoid of outer membranes. I never tried on brain mitochondria, but could finally get some results in liver mitochondria. I would assume that everything will be much more complicated in brain mitochondria because you start from a much lower amount of material and also you need to struggle more in eliminating all the myelin. How did you isolate brain mitos and how did you subfractionate them? 

Dr. Spinazzi,

Thanks for the quick reply. I apologize for the late response. The protocol for isolation is below:

1. Mice sacrificing. 

2.Brain samples are placed in a 9× volume of ice-cold IB (225 mM mannitol, 75 mM sucrose, 5 mM Hepes, 2 mM K2PO4, 1 mM EGTA (pH 7.2), 0.1% BSA). 

3. The resultant homogenate is centrifuged at 1,300×g for 5 min and save the supernatant.

4.The supernatant is layered on 10 mL 15% (vol/vol) Percoll (GE)

5. The samples are subjected to a centrifugation at 12,000 rpm for 10 min.

6. Carefully save the loose pellet and resuspend it to 10 mL cold mitochondria isolation buffer with 0.2% Digitonin. Stay on ice for 5 min followed by a centrifugation at 8,000 rpm for 10 min.

7. Save the pellet and resuspend the pellet in cold mitochondria isolation buffer w/o BSA and subject to another centrifugation at 8,000 rpm  for 10 min.

The final mitochondria is brown with a very small white fringe.

For fractionation: For the rupturing of OMM, I have tried various KCl concentrations, various concentrations of NP-40 and Triton-x or 5 mM Hepes.

I seem to be able to separate some OMM away from IMM and the matrix but seem unable to remove it all. If I increase detergent concentrations, I begin to break away IMM (I western blot for CCO or ANT for IMM and VDAC or mfn-2 for OMM). 

To separate IMM from OMM, I have used 35 % sucrose and a variety of centrifugation protocols without layers. I think 35% sucrose works well but I still have a lot of OMM marker with my mitoplast. Any ideas on removing all or most of the OMM from mitoplast without disturbing IMM?

Thanks

Dear Simon,

as mentioned I have never tried to do this specifically in brain mitochondria, which I use for other purposes, but I have just a couple of observations:

1) Be careful when using such a high concentration of digitonin in your crude brain mt fraction, it is possible that you already have broken to some extent mitochondrial outer membranes, which could compromise further subfractionations

2)KCL won't remove mt OMM but only peripherally attached proteins, and NP40 and TrX are probably worse choice than digitonin, which is anyway quite tricky to use (you can remove OMM, but the concentrations where you break IMM are very close)

3) A classical method is the swell-shrink protocol from Sottocasa, followed by mild sonication and sucrose gradient rate zonal centrifugation. Not perfect, but it works at least in liver mitos

A limitation there is that the amount of initial mitochondria that you need is quite high

Best regards

Marco

Central to most of the answers and methodologies is the notion that the mitochondrion is rather singular and not statistically distributed. Of course we all know that isn't true and yet we ignore it. The guiding principle for us was the marker enzymes, sulfite:cytoC ox.reductase and , say fumarase for the matrix space. Any swelling, sonication, detergent treatment should be time/intensity etc such that you know precisely at which point  these markers get released, both start and end points.There is no other way to have a control on the extent of degradation because all you get are enriched fractions any way. For instance, rat liver mitochondria have a centrifugal distribution starting with about 2000 xg to 12000 xg. An alternative method was to get a narrow distribution between , say3-4000 xg and proceed to subractionate it. The major force working against you is the distribution. Readymade methods...is really a pipe dream. Also methods that do not pay attention to osmotic pressure/rupture, unless deliberate, are suspect. Results could only be spurious.

 Dr. Spinazzi,

Thanks for the reply. I will try the Sottocasa protocol as you suggested. 

Are there commonly available markers for Drosophila mitochondria?

update: A list of some markers for fly cells is available in the following paper:

Article Differential centrifugation-based biochemical fractionation ...

Mitochondria isolation protocol?

I am looking for a reliable protocol for separating mitochondrial and measuring of cytochrome C release from human blood platelets. For this purpose, I collected platelets (5×108cells/mL), washed with cold PBS, and then, resuspended cells in permeabilization buffer containing 400µg/mL digitonin, 75mM KCl, 1mM NaH2PO4, 8mM Na2HPO4, and 250mM Sucrose, and incubated on ice for 10min. in next step, I centrifuged suspension for 5 min at 16000g, collected supernatant as the cytosolic fraction and then, lysed pellets in buffer containing 1% Triton-X100 for 30 min on ice. Ultimately, I centrifuged cell lysate for 15 min at 16000g and collected supernatant as mitochondria fraction. Also, i isolated mitochondia from blood platelets as described by Sareen D et al in their article (Mitochondria as the primary target of resveratrol-induced apoptosis in human retinoblastoma cells), but any extracted samples didn't work in western blotting and I did not had band.Has anybody any suggestion?

Dear colleagues, my best greetings to you!

Dr. Spinazzi raised an interesting technical question: how to separate outer membrane from the rest of the mitochondrion called “mitoplast”.

I would like to bring to attention several points regarding this issue and which one has to take into account. This is because the same method, which is good for the liver mitochondria, will not work with mitochondria from other organs.

1. Digitonin. This detergent works well as a detergent with membranes, which are rich with cholesterol. It works more or less effectively with many types of cells, but with others. from my experience, it was almost completely ineffective in disrupting prostane cancer cells. It will also not work with mitochondrial membranes because mitochondria have very little cholesterol. Digitonin is a detergent, therefore it is much wiser to avoid it when you want to disrupt cellular membranes to isolate mitochondria. The hypotonic method (described in my book “Practical Mitochondriology” and in the paper on prostate cancer) is much more reliable and safer.

2. The outer membrane has attachment points with the inner membrane at the sites where the adenine nucleotide translocase (ANT) is located. The more mitochondria contains respiratory complexes and thus more ANT, the more points of connections of the outer membrane to the inner membrane. This means that you can disrupt the outer membrane in liver mitochondria simply by causing the large amplitude swelling in hypotonic medium, or by overloading with CaPi in the presence substrates (permeability transition). But this method might not work with brain or heart mitochondria because much larger number of “stitches” – the contact sites.

3. The amplitude of swelling. This depends on the size of the osmotic volume of the matrix. From my experience the largest osmotic volume have rat liver mitochondria. Mitochondria from mice from any organ have significantly higher rates of respiration as compared with the rat mitochondria. Therefore even in the liver mitochondria mice have more proteins in the matrix and therefore smaller osmotic volume. To my estimation by 20-25% (see figure 1 in Panov et al., 2007. The PDF is attached). Mitochondria from the brain and heart have significantly smaller osmotic volume (compare Figs.1 and 3). This means that to cause large amplitude swelling to disrupt the outer membrane will be close to impossible in brain mitochondria.

4. There are several ways to cause large amplitude swelling: 1) by overloading with CaPi will cause relatively large amplitude in liver mitochondria, but not in brain and heart mitochondria; 2) addition of alamethicin causes large, I would say, maximal swelling; 3) incubation in hypotonic media with tonicity

Hi

Dear Panov

Thanks for your reply.

I also thank Dr. Panov, who gives extremely useful advices.

I experienced similar findings in several of these crucial aspects.

Kind Regards

We published in BBA sometime ago method for separation of outer and inner membrane of rat liver mitochondria. This method works well in our hands, better than Digitonin method. PDF of the same is enclosed. Hope this will help. All the best.

S. S. Katyare

Professor of Biochemistry

Thanks Mr. Spinazzi and mr. Katyare for your reply.

Hello Surendra

It has been a long time since we collaborated. This may help. In research back in 1969 on solubilizing a membrane-bound enzyme for study, the recipe required using digitonin. As much as I tried I could not get it to work. I contacted the author and he gave me a little unpublished step in this procedure that was the trick. Digitonin is very insoluble in aqueous solutions if you simply add it. You must first make your digitonin soluble and use it immediately with your membrane preparation otherwise it comes back out of solution within an hour or so.

The solubilizing of the digitonin occurs when you add the dried powder to your buffer, then place in a boiling water bath for a few minutes with rapid stirring, and then once the opalescent suspension clears, cool it immediately in an ice bath and begin slowly dispensing into your membrane prep at the concentration you need with stirring. Don't add more than is needed since the digitonin forms a complex with the membrane cholesterol and extracts it, modifying the stability of the rest of the membrane components.

I hope that helps. Many warm regards.

Joe Guth

Hi

Dr. Guth

Tank you for your reply.

I hope it works and is useful. Let me know how it turns out please.

Can someone point me to a good protocol that allows me to separate AND recover both IM and OM?

I'm looking to determine lipid levels in both membranes.

Dear Anna, that is a tough question. The easiest way to separate outer membrane from the rest of mitochondria is tow cause high amplitude swelling. But not all mitochondria can do it even in the presence alamethicin or hypotonic medium. Liver mitochondria swell easily, buy they have rather limited amount of many enzyme and functionally too far from other mitochondria. Mitochondria from cultured cells do not swell at all even after addition of alamethicin. Heart and brain mitochondria have too many "stitches" made by ANT-Porin contacts. The answer to your question depends on what type of mitochondria you want to study.

Unfortunately, cultured cell :)

I was also looking for reliable protocol but for brain tissue, anyone can recommend one?

I would also be interested in a protocol of separating and recovering IM and OM from brain tissue if anyone has experience with that.

Dear Kristina, for the brain mitochondria, we normally understand that the existing isolation methods usually provide us the synaptic mitochondria. These are derived from both pre- and postsynaptic elements. Functionally they are also similar. At least in major observed functions. However, what I know that my attempts to separate outer and inner membrane were unsuccessful. As well as to measure the volume of the matrix space. However, I think that you may change the approach by modifying the goals you have. Why it is so important for you to have separately outer and inner membrane? It would be interesting to discuss that.

Sincerely,

Alexander.

Dear Alexander, sorry for the late reply. Thank you for your insight. It is not that important that I separate inner and outer mitochondrial membrane. Since I'm new to mitochondria isolation methods, I came across different data (e.g. from liver tissue and cultured cells) that people are separating those membranes and I simply thought I might gain an additional level of insight. If it is that problematic I won't even attempt that, at least not for now. Just isolating synaptic mitochondria is fine.

I would gladly help you find your way in mitochondrial studies. I have 55 years of experimental work with mitochondria from different organs and tissues. If you let me know what type instrumentation you have, we could discuss from what function to start.

As a matter of fact, to isolate and to study brain mitochondria (BM) is much easier than, say liver mitochondria (LM). On the contrary, you may have a lot of LM just in one hour with minimum equipment, but to study LM from the functional point of view, is extremely difficult. This I explained in my book "Practical mitochondriology". You may copy it from my ResGate site. The hard copy you can buy on Amazon just for, I think 5 dollars. To study mitochondria from cultured cells is extremely difficult and expensive. You need the top level of culture facility to grow up cells without antibiotics. Most people do not have such facilities, and therefore use antibiotics, which are toxic to mitochondria. The are other reasons, which make cell culture approach obsolete from the point of view of physiology.

If you wish, we can continue our discussion. I am interested that young researchers would use some of my new approaches to study mitochondrial physiology. On my site you can find some of my latest papers that describe new paradigm to study mitochondrial functions.

"You need the top level of culture facility to grow up cells without antibiotics. Most people do not have such facilities,"

Imho, you don't need any special facilities to grow cells without antibiotics, and any lab having a properly maintained cell culture hood and cell culture incubator will do just fine. Though, it does require being more careful and meticulous to maintain sterility to avoid bacterial contamination, so antibiotics are often helpful to ease this up.

Dear Michael, I agree with you that you can grow up cells without super-duper special conditions. However, this you can do, if you grow up cells in relatively small quantities. However, if you want to ISOLATE mitochondria from the cultured cells you MUST have huge amount of cells. When I was isolating mitochondria from the patient's lymphoblastoid cells, I used sometimes 6-9 liters of cell suspensions. The amount of mitochondria in cells varies hugely between the cell lines. For isolation of mitochondria from the cultured prostate cancer cells (I used three different cell lines, plus control cells from normal prostate) I had to have at least 15-20 largest flasks. You have to keep in mind that when cancer cells approach confluence higher than 60% (that happens usually after 12-20 hrs after changing medium at around 50% confluence) mitochondria initiate apoptotic changes. First, you see that membrane potential is low, whereas "normal" potential in prostate cancer cells is higher than 200 mV. This is a specific property of these type of cells. Antibiotics, like streptomycin, make mitochondria in cells incapable of respiration, penicillin is known as an inhibitor of protein synthesis. Thus you cannot talk of any role of mitochondria in anything, if you use antibiotics. In addition, there are other properties, which acquire cells after transformation. For example you cannot make mitochondria swell, even after addition of alamethicin. They change metabolic phenotype, etc. It is much more informative and closer to normal living conditions to study incubated tissue slices. This is particularly important in case of brain, where astrocytes metabolically support neurons.