Dear Jathupon

When we used Olygomycin to inhibit the complex V (ATPase) we did not isolate the mitochondria, we incubate our primary cell cultures with Oligomycin at different times and concentrations. In your case, i don´t think the problem is due to mitochondrial isolation or solubilization since cases 2 and 3 show constant rates. The protocol you use seems to be ok. Did you perform replicates within the same experiment or you find the variation among different experiments?

Hi,

Please find attached a reference for mitochondria isolation. The procedure mentioned therein is gentle and suitable for such assays. The problem you are facing might be due to differential solubilization efficiencies, causing the variation. I would suggest use of permeabilized mitochondria ( a classical way ), instead of solubilization. I have also attached herewith a reference--the same procedures of permeation can be applied also to mitochondria, as done in original references.

Contd with attachment--

Dear dr. Nakhonsri,

I do not have any experience measuring complex V activity in LDL but i can give you our method for cultured fibroblasts and this method works perfectly. I described the isolation procedure for mitochondria from cultured fibroblasts in: Janssen A et al., Clin. Chem. 2007;53:729-734. We described the complex V assay in: Morava E et al., Am J Med Genet. 2006;140A:863-868. Using this assay you can measure complex V activity with 0.02ml of the mitochondrial suspension isolated from cultured fibroblasts instead of muscle mitochondria. I think these methods can also be used for LDL.

Best regards,

Antoon Janssen

You might want to check out a reference by David Andrews on the use of a pressure device to isolate intact mitochondria from mammalian cells. This involves a sudden release of pressure through a valve on a high-pressure 'bomb'. We used this many years ago and it provided a high yield of intact mitochondria. Not clear if this will help but it works. .

If you can check out a reference by David Andrews on the use of a pressure device to isolate intact mitochondria from mammalian cells

Hello, I have isolated mitochondria from >10 cell lines and primary tissues using various methods. You will have to do some trial and error with isolation buffers and isolation procedures to see what works best for your cells.

The first key to getting healthy, intact mitos is using the correct isolation buffer- different tissues have varying needs so there are some subtle changes depending on the cells/tissue used, but regardless you need a sugar source (usually glucose but trehelose works too), a kelator (EGTA or EDTA), and a buffer for pH (Tris-HCl, MOPS, etc.). I generally start off using a traditional mouse liver mitochondria isolation buffer when trying a new cell or tissue type since it is very universal. Then, if you don't get good results you can start to change components. There are specific recipes for brain/neuronal cell mito isolation, kidney cell mitos, differences between rat and mouse tissue, and the list goes on and on. Here is my mouse liver mito recipe (250mM sucrose, 1mM NaEDTA, 10mM Tris-HCL, pH 7.4 and then filter).

Once you have isolated mitos, if you want to use them in a functional assay, such as ATP turnover, delt psi, ETC inhibitors, cytochrome c release, etc. then you need to then dilute your mitos into an experimental buffer that will promote respiration and membrane potential. Typically I isolate the mitos as a concentrated solution, run a protein assay so that I can normalize all my assays from day to day withe same amount of total protein (in this case mitochondrial protein) and then dilution the mitos down to 0.5mg/mL with an Experimental Buffer (KCl 125 mM; TRIS/MOPS, pH 7.4, 10 mM; Glutamate 5 mM; Malate 2.5mM; K phos, pH7.4, 1 mM; EGTA/TRIS10 microM). Again, some changes can be made to experimental buffer for optimization.

For isolation technique there are several choices: The nitrogen bomb that was referenced above from David Andrews' lab works fine but you have to buy the bomb and while it is fairly easy to use it takes some getting used to because when the pressure gets released at the end if you are not careful your sample can go all over the place which results in loss and/or frothy foamy solution. Traditionally, mitochondria isolation is done with a Dounce homogenizer (tight fitting pestle and glass vessel), whether using cultured cells or primary tissues from an animal either by hand or attaching the pestle to a drill. However this also requires specialized equipment that you may not have, however it is a very universal way to isolate mitochondria so if you plan to do many isolations then it may be worth buying. Alternatively, you can use disposable syringes and needles- pull up the solution of cells using an 18 gauge needle into the syringe and then change needles to something with a smaller bore such as 26 or 27 1/2 to push the cells out and then repeat between 5 and 15 times depending on the cells being used so some trials need to be done. Any of these methods are using force to break open the cells and release mitochondria so they then can be isolated through differential centrifugation.

For centrifugation, a low speed spin of around 500 - 800 x g, 4C, 10 minutes is done once or twice to get rid of any heavy cell membranes, unbroken cells, blood if you are using a tissue, then a 10,000 x g, 4C, 10 min spin to pellet mitochondria (note that this is a crude isolation and you will have some ER attached which is OK for functional assays but not for import assays or isolation of mtDNA- in those cases you need to do a high-salt wash to get rid of ER).

Sorry for the length, I hope this helps and feel free to email/message me if you have questions.

This a good start to find a working protocol.

http://books.google.ch/books?id=o8guY4Twpe4C&printsec=frontcover&dq=subcellular+fractionation&hl=de&sa=X&ei=tEGCUtLxBsjdtAbvpYG4Dw&ved=0CEkQ6AEwAg#v=onepage&q=subcellular%20fractionation&f=false

Please have a look to this paper

please have a look to this paper.

I suggest to assay ATP synthase in intact organelles (cell homogenates) as in Gagliardi S et al Bichem. Mol. Biol. Inter.

A dumb question: why do you expect to see a difference between (1) and (2) with SOLUBILIZED mitochondria? I would expect them to be pretty much the same. What is it that I am missing?

I am using a wonderful protocol to isolate intact mitochondria from cultured HeLa cells (authors also tested with fibroblasts, liver, muscle etc.). It is easy, no unusual reagents, quick.

Frezza et al., Nature Protocols 2007. (attached)

Although I don't do ATPase activity assays, they do also describe assaying oxygen consumption (etc.) with the purified, intact mitochondria isolated at the end of this protocol. Could be done.

I'd suggest trying this assay, though. The most difficult-to-find reagents are EGTA and sucrose (both very common), prep time is quick, experiment time is minimal.

Hello Thanks everyone for sharing.

Dear Dr.Ignacio Rego,

Well, I'm using oligomycin in order to specifically observe F1Fo ATPase activity not the sensitivity to oligomycin itself. I don't think I could add oligomycin into the culture.

About doing in replication, yes I did and on the same day (same preparation) outcome are mostly similar.

Dear Dr. Diksha Dani,

I've been roughly through the article you shared.

It's plant's organelle isolation isn't it?

Apart from that, with other respiratory enzyme assays, using sodiumcholate to solubillize works fine. I guess it's because F1Fo ATPase is way too fragile than the others. Hence, I'd be looking for, if any, specific protocols that works with oligomycin sensitive activity assay.

However, I couldn't agree more that it could be from solubilization but I've not proved yet. May be, I'll compare solubilization on the same mitochondria on the same day to see variation from this. For the classical way, I found a simple way that I could try that is to repeat freeze-thaw. I'll let us know what comes after.

Dear Dr.Antoon Janssen

First of, it's LCL(Lymphoblastoid cell lines) not LDL(discovering ATPase activity in LDL would be very interesting though!) which is transformed B-cell by EB virus. About the publication you mentioned, they're using freeze-thaw method to solubilize mitochondria. I start thinking this is the real source of problem I've encountered.

Dear Dr.David Kessel and Dr.Asenin Betr

That's fancy. Sounds fun. But I guess my supervisor's not gonna be happy to buy new equipment if it doesn't show greater significant usefulness. I'll try with the conventional one first.

Dear Dr. Victoria Del Gaizo Moore

Long post is not bothering at all. Instead, I'm really grateful for your afford sharing me high detailed information.

So you've isolated over 10 mammalian cell lines, that's wow.

I'm so lucky to meet up mitochondrial veteran here.

Well, from your experience, have you ever dealt with blood cells like lymphocytes or lymphoblastoid cells? I wonder if I'm using the correct buffer. Its content is 220mM mannitol, 70mM sucrose, 5mM MOPS pH7.4. Tell you the truth, I thought all isolation buffers are the same so I got the recipe from other mitochondrial lab which use C.elegans as a model. Poor me.

Then you talk about experimental buffer, I guess you mean solubilization buffer. Well, I used KME as an experimental buffer. It comprised of 1 mM Tris, pH 8.0, with 50 mM KCl, 1 mM MgCl2, and 1 mM EGTA. Once again, I borrowed the recipe from C.elegans lab.

About breaking the cells, as I mentioned I used sonication method. Do you think it's gonna be problematic? This one was successfully used to isolate mitochondrial proteins from fibroblast cells. Could it be harmful to my enzyme? By the way, I found some article using sonication to breakdown mitochondria for OXPHOS enzyme assay including CV. I think it's gonna be fine with this too. What do you think?

And come to centifugation, here I found that I washed mitochondria so many times. Indeed, this one is the protocol our lab used for mitochondrial proteomic study which needs quite pure mitochondria. I will try reduce speed, step and duration to preserve as much live mitochondria as possible.

Thank you very much for the length.

I wish to contact you again when anything get updated.

Best Regards.

Touche.

Dear Dr.Alain Schilb

The book looks good but I feel dumb and don't know what to look for.

The question is quite too specific so I'd prefer reading through out the book someday to give myself more information/knowledge to deal with mitochondria in the future.

Thanks by the way.

Best regars.

Dear Dr.Alain Schib

The protocol is very useful. There're many tricks and trouble shootings.

It's gonna be very useful for my mitochondrial isolation from fibroblast.

By the way, our laboratory doesn't have oxymeter. I have no choice but to set up colorimetric assay which depends on substrate accessibility to the enzyme.

Thanks for sharing though.

Dear Mikhail Alexeyev

Do you mean that solubilization by sodium cholate would've killed all enzyme, am I right? May be I'm using confusing term of solubilization. I mean to flip mitochondrial membrane out in order to make membrane bound enzyme to expose to added substrate.

And the reason why I expect to see (1) differ from (2) is that the assay reveals ATPase activity and not specifically describe ComplexV activity. So, I used inhibitor to see how much it change after I put inhibitor into the assay. As much change as it has more ComplexV activity. Am I answering your question?

Dear Dr.Natalie Marshall

As I told Dr.Salvatore Passarella, we don't have oxymeter here. So, I have no choice but to break down mitochondria.

Thank you for sharing though.

Best regards,

Touche.

Dr. Nakhonsri,

Yes, I also lyse my mitochondria after isolating them. I use the Frezza et al. technique to get rid of the cytosolic proteins (etc.) and isolate only mitochondria, then I lyse them anyways, usually for Western blot. So if you're using sodium cholate anyways, it would still work with this technique, you'd just need an oligomycin ATPase assay for afterwards.

Best.

Dear Dr.Marshall

When I do Western blot, I use lammali's buffer which contains SDS and b-mercaptoethanol to lyse mitochondria.

I don't think enzyme would survive after all.

Are you using different buffer?

Yes, I have isolated from blood cells- primary chronic leukemia cells and primary acute leukemia cells from patients and several acute leukemia cell lines. The buffer recipe I gave in my first post is what I used for all of those cells. I worry about the mannitol and MOPS in the buffer you are using- I have used manitol when isolating mitos from rat liver but have not had success with it in any other mito isolation; generally sucrose is used. For the solubilization buffer (what I call experimental buffer) I worry about the KCl and MgCl2 in yours; I use KPhos which is a balance of K2HPO4 and KH2PO4 since the chloride ion may be problematic keeping the mitos isotonic. I have use sonication but only when I wish to break open the mitos and make mitoplasts and mito ghosts (outer membrane only and inner membrane and matrix only). This may be OK if you are looking at ETC, as I have seen this before in papers. I have done assays similar to the ones you are describing- adding oligomycin or other mito uncouplers and/or adding NADH or other electron sources such as succinate or adding ATP and ADP (I designed a teaching lab experiment for my advanced biochemistry class that utilizes mouse liver mitochnodria and they learn about ETC inhibitors through different additions). In these experiments we use TMRE to look at changes to mito membrane potential as a read-out for any ETC/respiration changes. You could do the same if your plate reader can detect fluorescence.

I don't have a Clark electrode or oxymeter but have recently assayed mitochondria for oxygen consumption using a fluorescent plate reader along with MitoXpress from BMG Labs and it worked really well- you use mineral oil on top of your reaction so that no oxygen is exchanged with the air once your reaction starts.

Dear Jatuphon Nakhonsri,

Oligomycin blocks proton translocation through mitochondrial ATPase (complex V) from the intermembrane space into the matrix. When you solubilize your mitochondria, with sodium cholate, proton gradient is dissipated. Moreover, the inner mitochondrial membrane is destroyed and likely electron transport chain no longer functions as it does in intact cells. The way I understood your assay, you follow NADH oxidation by complexI, and, provided enough coupling, inhibition of the complex V with oligomycin can indeed slow down NADH oxidation giving you some difference between (1) and (2). However, detergent (sodium cholate) treatment should efficiently uncouple NADH oxidation from ATP production, so that I would not expect any specific effect of oligomycin on NADH oxidation. Therefore, my question: why do you expect to see a difference between (1) and (2)? Can you provide a source of the protocol that you follow?

Dear Dr Moore,

Thank you in advanced.

I'm not sure about mannitol and MOPs. Do you know what does mannitol do in isolation buffer. I found that it just gives one extra NADH when oxidized to fructose.

However, I'll try altering buffer from mannitol/sucrose to sucrose buffer as you suggested. About the solubilization buffer, the sodium cholate protocol suggest that kind of buffer. Here, I suspect solubilization process could've mattered the variation of the result. Therefore, I will turn to conventional method that is freeze/thawing method which have been done in 20mM KPi buffer(KH2PO4).

Looking at membrane potential to track mito-respiration is a good idea. Actually after seeking direct oligomycin sensitive ATPase activity, I will go for mitochondrial membrane potential.

It may be a stupid question. But, why do we need to follow oxygen consumption. Doesn't it represent cytochrome C oxidase activity(CIV) alone by seeing oxygen consumption?

Thank you again for further information.

Best regards,

Touche.

Dear Dr. Alexeyev

Here is the protocol I'm following(see attached file Page. 68-84)

The assay aims to monitor ATPase activity of F1Fo ATPase not ATP synthase activity. Instead of making ATP, I encourage the enzyme to utilize ATP and reverse proton translocation. In the assay, ATP will be regenerated back by Pyruvate kinase that turn PEP to pyruvate. Later, pyruvate will be reduced by lactate dehydrogenase using NADH as a reducer. As long as NADH oxidized, OD 340 drops.

Taken together, the assay doesn't need intact mitochondria and cooperation of other OXPHOS enzymes. Indeed, I have to inhibit CIV with KCN to prevent forward reaction(ATP synthesis) by functional OXPHOS.

Best regards,

Touche.

Other researchers in our lab have used CHAPS (2% I think). However, I do not do enzyme studies after mitochondrial lysis, just a simple lysis in Laemmli for Western blots, so we're doing different things at that stage. Our protocols will be different there.

You initially mentioned that mitochondrial isolation might be one of the issues you're experiencing, so I am suggesting you try this technique, which is quick, easy, and cheap to isolate mitochondria. At the end of the suggested protocol, you have intact and functional mitochondria, which are useful for an array of experiments, such as perhaps ATPase assays.

Dear Marshall,

Surely, the protocol you put there will be very helpful.

It contains tips, critical steps and trouble shootings as many as I've ever seen. I plan to follow it when I shift to do mt isolation in fibroblast in the near future.

Thank you very much.

Best,

Touche.

Dear Jatuphon Nakhonsri,

Thank you for the reference. Do you know whether this protocol has been validated for mitochondrial preparations? It seems that in order for this assay to work, the rate of ATP hydrolysis by complex V should be at least on par with non-specific ATPase activity. Also, ideally there should be little contribution from nonspecific NADH dehydrogenases/ NADH autooxidation. Typically, nonspecific NADH dehydrogenases present a challenge when assaying Complex I activity. In those cases, people measure rotenone-inhibitable (specific) and total NADH oxidation. Do you know that sodium cholate solubilization preserves both ATPase activity and its oligomycin sensitivity?

Great, glad it was helpful! It's a great protocol. Best of luck.

Dear Dr.Alexeyev,

You've got good points.

Let's say, I don't have information in my hands whether how much F1Fo ATPase is, comparing to non-specific ATPase. However, NADH coupled ATPase activity assay is already set up conventionally for mitochondrial ATPase activity assay.

About sodium cholate, it's a big pit fall I made for myself, I guess.

I started practicing the assay together with other student who's assaying CI-CIV of C.elegans. I didn't concern the fragility of CV before, therefore I used their solubilized mitochondria sample for CV assay and it seemed working fine. But later when I changed to do the assay on my own sample, LCL, I started having problem with too small difference in oligomycin sensitive activity. I tried to adjust the amount of mitochondrial protein, substrates and enzymes till it get the best of two fold rate different in oligomycin sensitive activity.

Later on, I tried to repeat it with the same method. I found it's constant in non-specific ATPase and spontaneous rate but not in oligomycin sensitive rate. So, I wonder what could make my F1Fo ATPase "die" in sometimes and not in the other times. After I posted here and got some suggestions, I turned back to review literature and I don't find any of them use sodium cholate to prepare mammalian mt-sample for F1Fo ATPase assay. Rather, they use frozen/thawed method.

Not only that, some small other pitfalls that may not have much deal with the assay are discovered here too. For instant in the mt isolation process, I washed it by centifugation too long and too often. The protocol I used to isolate mt at the start is the one for mt proteomic study which needs highly purified mt and not care about live mt.

On this Friday, I plan to isolate mitochondria with lower speed, time and one step of purification. On the assay, I will use freeze/thaw method to break up mitochondria compare to cholate solubilization. Anything do you want to add up?

Dear everyone,

After I modified mt isolation and solubiization, the assay is now working well.

What I found here are

1. the first protocol I used for mt isolation is for high yield and less contamination of cytosolic protein which is good for proteomics but not for enzyme. After I altered spin speed; 20,000xg to 15,000xg, duration; 30 minute to 10 minute and reducing one washing step, I got less yield(2 fold) but, may be, healthier mitochondria.

2. Comparing between sonication and using syringe to break down cells, using syringe gives slightly more oligomycin sensitive rate per ug protein but with lower abundance.

3. Freeze/Thaw method obviously increase oligomycin sensitive rate compared to sodium cholate solubilization. Non-specific ATPase and spontaneous rate are not altered by freeze/thaw.

Next is to repeat it again.

Thank you everyone for your advice and valuable suggestions.

Special thanks to Dr.Moore who guide me a lot and Dr.Alexeyev for his critical questions.

All the bests to you all!

Touche.

I feel what you may try is to simply replace Mannitol in the buffer. Use a low concentration phosphate buffer. I would also recommend that you check the sodium cholate that you are using. It is no doubt a text-book method to use this, but try using sodium desoxycholate. May be your problem could be solved by these simple modifications.