VDAC (outer membrane), COXIV (inner membrane), CytochromeC (intermembrane space), TOMM20 (outer membrane) are all good mitochondrial markers.

You can use VDAC or COXIV

Thank you very much.

A good marker is TOMM20. As it is localyzed in the outer mitochondria membrane it is also a control of the integrity of your isolated mitochondria

You could also use GAPDH. There are very good and reliable antibodies against it.

I usually use TOMM20 and works very well.

As Ivan Zaja say, you can use VDAC or COXIV, there are very good antibodies against these proteins. Do not use GAPDH, this protein is located in cytosol and in mitochondria.

Maybe you can use a total protein stain as a loading control, instead of just one particular protein.

U can Use Cox IV ab. I used that for my experiments

http://www.ncbi.nlm.nih.gov/pubmed/21623955

hi,

cytochrome oxidase should be a good marker of well purified mitochondria as well. I used it years ago and worked very well.

Alexandre if what Diogo wants is just a loading control, in the same way in which actin is used for whole cell lysates, he will not have such contamination problems with the citosolic GAPDH once the mitochondrial fraction is purified, but if what he wants is a specific marker for the mitochondrial fraction in order to know the purity of his mitochondria isolation method then I agree the any of the other proteins proposed here are indeed good candidates.

The succinate dehydrogenase can be a good marker, we use anti SDH-A.

VDAC (outer membrane), COXIV (inner membrane), CytochromeC (intermembrane space), TOMM20 (outer membrane) are all good mitochondrial markers.

The proteins Benoit Vanderperre stated here are indeed good markers. However, I would also try a plasma membrane marker, because mitochondria are usually contaminated with plasma membrane if you don't use some kind of gradient purification.

I suggest to use TOMM20, instead of cyt C due to not uniform distriubution, since some cells could be apoptotic; this depends on the cellular stress that you induce.

Heat shock protein 60 works brilliantly. It is located exclusively in mitochondria. the antibody from stressgen is very good- one of the most effective antibodies we use.

VDAC is great control for mito proteins, Abcam's website even has a list of protein controls to use for nuclear, mitochondrial and total protein lysate. Another option is to stain the blot (if using PVDF) with Ponceau S Red.

The most common source of cross contamination in mitochondrial preps is endoplasmic reticulum membranes. Relevant antibodies in this case include those anti- calcineurin, calreticulin, IP3 receptors, etc.

I suggest to use TOMM20, instead of the common cyt C; the latter could be either apocytochrome c or the complete form and mislead the result (this is a very fine tuning for WB assay); furthermore the cell could be apoptotic, but depends on the study that you performed.

It may depend on the type of mitochondria and especially by the method of purification. If you are sure to have mitochondria intact and not contaminated from the cytoplasm may be convenient to use VDAC, CytochromeC (intermembrane space), TOMM20 (outer membranes)But you can use the mentioned proteins also together with a total protein mix as loading control, but placed in two different lanes.

We've been having good results using porin (outer mitochondrial membrane). This also allows you to determine that your mitochondria are intact after isolation.

TOM20 or VDAC or PORIN will be good markers to use

Our lab often uses pyruvate dehydrogenase E2/E3 antibody from abcam (http://www.abcam.com/Pyruvate-dehydrogenase-E2-E3bp-antibody-13G2AE2BH5-ab110333.html). The antibody works well and PDH is a good mitochondrial marker. Good luck!

For stating purified mitos, all markers have been suggested here. However, we also have to exclude contaiminations, so you need test actin/calnexin/lamp1 etc bloting as well

Use coxIV

TOM20 and TOM40 are both good. Good protein markers to control the purity of your preps. There is small amounts of actin in mitochondrial matrix though. See: "Purification and immunohistochemical study of actin in mitochondrial matrix." (PMID: 2346501) and "Actin and myosin contribute to mammalian mitochondrial DNA maintenance." (PMID: 21398640)

All above are good suggestions. As we used to work with unusual mitochondria I needed to use a few others that have shown to be useful for 'standard' mitochondria as well: the TCA cycle enzyme succinyl-CoA synthetase is quite conserved and the mitochondrial chaperones (Hsp60 and Hsp70) are good too. Be ware, there are cytosolic and ER versions of Hsp70 too so make sure you use the correct one. Hsp60 is highly conserved (like mHsp70) and commercially available.

TOMM20 and COXIV are good markers. I suggest to use some ER proteins as purification quality control (calnexin, IP3 receptor).

I have successfully stained mitochondria with NRF1 and succinate dehydrogenase, yielding exquisite immunofluorescence. Both also work well by immunoblot.

Thank you all.

I prefer prohibitin (PHB1) it has a higher molecular weight than COXIV.

Also remember to blot with a cytosolic protein (such as b-tubulin) as control!

Use cox IV antibody or COX I also csn be used

Don't use VDAC, as it is clearly present in the plasma membrane as well.

We use BD Biosciences HSP60 and MnSOD antibodies. Beware, though, that HSP60 gets highly enriched in mitochondrial preps (we have to use 1:40,000-1:100,000 dilutions of primaries).

Prohibitin is one of the best markers for mitochondria preparation

Be aware that Prohibitin is also found in the nucleus and VDAC as mentioned above is also located in plasma membrane. COXIV probably is a better and more specific choice. Simply, If your preparation is positive for Cty C and negative for actin then you probably have a pure preparation. Good Luck.

The best control is porin if you want to use it as a loading control. If it is only to determine the presence of mitochondria, whatever indicated by Benoit.

As many "control" proteins may change depending on treatment, I prefer to strip the membrane and do a Coomassie R-250 stain for all the protein on the membrane and then use a representative band as a control.

As Gisela indicates, this is a good option to demonstrate the total protein loading in each treatment. We also use red ponceau that can be scanned and quantified to be used for total loading corrections. Membranes can be stained with red ponceau before being used for immunostaining. Red Ponceau is used dissolved in 1% acetic acid and is gone after incubating at neutral pH.

It depends on the type of samples (i.e. treatments/disease etc..). You have to check that the mitochondrial loading control that you choose is not influenced by your experimental condition (obviously COX will not be a good loading control if your treated sample have a cytochrome c deficiency). In general HSP60, TOMM20 are good loading controls, but others may be good as well depending on which conditions you are testing.

Hello, you can use anti-VDAC or anti-COXIV - Good Luck!

On this same topic, anyone could recommend a reliable antibody to detect a mitochondrial protein coded by the mitochondrial genome?

Hopefully something that is cross-species reactive...

Molecular Probes (Invitrogen) have reliable antibodies against both, nuclear and mitochondrial DNA encoded proteins. They use to work properly.

Stripping western blot membranes is not at all a controlled process and you will certainly loose proteins, therefore affecting any measurement you make afterwards. If you want a yes/no answer, stripping might be ok (although you will loose signal and possibly some low amount bands). However, for any serious quantification, stripping is simply not ok.

Hi Alexandre Costa,

How would you suggest to do the control?

If you are arguing that a control protein should be assayed at the same time as the target protein then how can you be sure that the signal your looking at is from your target protein and not from the control, especially in cases where you have a protein that is close in size to your control? Also, different antibodies may require different conditions.....

It is true that there is some loss of protein when stripping but one would have to hope that it is an even loss across the membrane. I have great success with stripping and staining with coomassie blue R-250 as a control.

Gisela

Hi Gisela! This is an interesting discussion!

First of all, let´s make a distinction here. "Loading control" is different than a "control protein" in your sample. Loading control can easily be done with Ponceau (or Coomassie) staining, as you and others mentioned. However, I understood Diogo is asking for an internal control for his samples, such as a housekeeping gene.

As I said, stripping is ok, but only for qualitative purposes. I used it myself several times. However, as you said yourself, you cannot guarantee a homogeneous process across the membrane, therefore it is not useful for quantification. Also, you would not be able to compare membranes from different experiments.

What I would suggest in the case you mention (proteins with similar size and/or antibodies requiring different conditions) is to run several replicates in the same gel, cutting the membrane lanes and testing each lane with a distinct antibody. For your loading control, I would do the same but cut the membrane horizontally (instead of vertically), and then incubate each piece of membrane separately. For example, check out figure 8 of the attached paper. We tested several proteins (same PAGE, same membrane) by cutting the membrane horizontally and incubating each piece of membrane with the corresponding antibody. To know where to cut, we would run a pre-stained marker, which is easily visualized on the membrane.

I hope I was clear. If not, please let me know!

A word of caution regarding actin. I would not assume that this is a good marker of the purity of your mitochondrial preparation. A lot of research indicates a tight association between mitochondria and cytoskeleton, particularly actin cytoskeleton. There is also some evidence of intramitohondrial actin localization. See, e.g. Nucleic Acids Res. 2011 Jul;39(12):5098-108.

Actin and myosin contribute to mammalian mitochondrial DNA maintenance.

Reyes A, He J, Mao CC, Bailey LJ, Di Re M, Sembongi H, Kazak L, Dzionek K, Holmes JB, Cluett TJ, Harbour ME, Fearnley IM, Crouch RJ, Conti MA, Adelstein RS, Walker JE, Holt IJ.

MRC Mitochondrial Biology Unit, Cambridge, UK.

Hi Alexandre!

Indeed an interesting discussion (my apologies to those of you who are looking for a straight answer) and your suggestion is good. However, I think it is important to discuss what a "housekeeping" gene really means. In my opinion there are no genes that can be trusted to be expressed at the exact same level in all cells at all times. In my case, CD40 signals alter just about any "housekeeping" gene I test. For example, actin is highly upregulated by CD40 signaling in the cells I study. Therefore, if I were to use that as an internal control, my actual results would be completely distorted and not reflect what is really happening. While Ponceau or Coomassie is great for a loading control, I would argue that a representative band from such a stain could also be used as an internal control. If we see the same trend each time we perform a biological replicate and the loading control/representative band indicates that there are no big pipetting errors, we can believe that a real biological response is studied. While I still agree that there is loss of protein when stripping a membrane (and I wouldn't recommend doing so repeatedly unless it can't be avoided due to precious samples being limited) I would still argue that the loss would be fairly uniform across the membrane (just like we trust that the transfer of protein from the gel to the membrane is uniform) and therefore a stripped and Ponceau/Comassie stained band can serve as an internal control.

Best,

Gisela

I use anti-COXIV and it works well for me.

I would not recommend using anti-cytochrome c, because it is released into the cytoplasm upon the induction of apoptosis. Therefore, if your cells are undergoing apoptosis, your control won't let you distinguish between mitochondrial and cytoplasmic fractions.

I'd also suggest using an ER control (e.g. calnexin) since these fractions can contaminate each other.

Best of luck!

Just wanted to point out, calnexin (ER membrane protein) has been mentioned several times as a possible control for ER contamination in purified mitochondria, however calnexin is also found in the mitochondrial membrane:

"Palmitoylation is the switch that assigns calnexin to quality control or ER Ca2+ signaling." PMID 23843619

I would use VDAC or Cox....but also go for coomassie staining. Depending on the type of desease/surgery/treatment mitochondrial proteins might change....for example, following contusion spinal cord injury, with in 24 hr PDHC and cytochrome c oxidase reduced significantly.

So, it is alwase safe to check with coomassie staining.

Best luck!!!

We generally use COX or cytochrome c. Luck!

I know this discussion is already a bit older but I wanted to give a comment anyway :)

No matter what you use for a loading control, housekeeper or Coomassie/Ponceau or even simple protein labeling, it leaves you to hope or guess that everything was pipetted correctly, transferred evenly and washed off evenly. You do not know because you can not monitor it. That was the reason why we added additional internal standards, to monitor loading and labeling efficiency as well as differences in protein concentration. We wanted to know, not to hope anymore. Now we have a system using the total protein (fluorescent labeled and co-detected with the target) to normalize the target + the added internal Smartalizer to normalize the total protein itself. We think that this gives now the opportunity to get the most reliable data out of your Western blot, no matter what may or may not happen on the long way to target detection.

If our system sounds interesting for you, please send me a direct message.

Best wishes, Kristin