Mitochondria Isolation Kit for Cultured Cells (ab110170)

Red blood cells do not contain mitochondria so PBMC isolation is recommended.

Ab110170 can be used to make all of the fractions you need. After the Spin 2 step in the protocol, the supernatant is the cytosolic fraction. The mitochondrial pellet will be largely intact mitochondria. In order to rupture the mitochondria, you can use a mechanical method (homogenization) or use a detergent like 10% lauryl maltoside ab109857- https://www.abcam.com/10-lauryl-maltoside-solution-ab109857.html

With regards to your questions:
1. What is a cell lifter? Can cells be detached by regular trypsinization (3 min at 37 degrees)?
A cell lifter is a tool used for scraping cells from dishes and plates.
Such as this one available through Sigma: http://www.sigmaaldrich.com/catalog/product/sigma/cls3008?lang=en®ion=US
Cells can however be detached by regular trypsinization, but make sure the trypsin is neutralized after the trypsinization, i.e. adding media.
2. "Freeze the cells and then thaw them in order to weaken the cell membranes" For how long should the cells be kept at -20?
Freeze /thaw cycles are usually done by dipping a cell vial into liquid nitrogen untill the cell pellet turns white, then dip the vial into 37C water, untill cell pellet turns back to its original color. It takes around a minute for each step.
After the cells have undergone the freeze/thaw cycle, the pelleted cells should be frozen at -80C into a dry pellet at least overnight.

There is no detergent in the buffers of the mitochondria isolation kit ab110170. The mitochondria membranes will be intact.

However, the lab warns that the preparation is not 100% pure and there could be plasma membrane or a small amount of other cytoplasmic organelles.

Western blotting, particularly using chemiluminescence, is not very quantitative so it’s a little hard to say what proportion of LDH is in the mito fraction but clearly there is some there.

Increase the number of wash steps of the mitochondrial pellet so repeat step 14 two more times.
Nuclei and unbroken cells are in the pellet of step 8 (not SN1)– this could be probed for the nuclei proteins but be sure to completely take up the pellet in sample buffer. Also the presence of unbroken cells will likely provide contamination.
Mitos specifically spin out about 12000 g, so the first SN after spinning out the mitos in step 14 will contain the smaller organelles and cytoplasm.
Steps - Cells are ruptured, then large organelles (including nucleus) and unbroken cells are centrifuged into a pellet and supernatant is collected in SPIN1, this rupturing is repeated on the pellet to maximize yield and generate super from SPIN2 which is polled with supe from SPIN1. Mitochondria are in the supe and are traditionally collected by centrifugation at 12000g this is done in Step 14 – in this case I would recommend washing this pellet to remove contaminating soluble LDH.

Thank you for contacting us and your interest in our products. I am sorry for the delay in getting back to you.
I have consulted with the lab over the suitability of the two isolation kits, ab109719 and ab110170, in regards to your customers problem with plasma membrane contamination. They have confirmed the following:
When ab109719 is used, the plasma membrane and mitochondria co-fractionate together, so ab109719 would definitely not recommended to isolate plasma membrane-free mitochondria.
I would recommend to use ab110170 as a first step to isolate the mitochondria. However the mitochondrial fraction prepared using this kit will still heavily contaminated with the plasma membrane. As an example, see the attached Western blot analysis of fractions isolated from mouse heart homogenate (MHH), including isolated mitochondria and supernatant 2, using a similar method (ab110168, a tissue version of ab110170) with plasma membrane marker (Sodium Potasium ATPase).
In order to isolate plasma membrane-free mitochondria, gradient centrifugation (for example percoll/metrizamide, sucrose, ficol) must be used on the crude mitochondria fraction. There are variety of protocols to achieve this, my favorite: Methods in Enzymology, vol 182, 203-225 (1990). If you are concerned with plasma membrane contamination and want to follow mitochondria as well as plasma membrane marker, I would recommend to use the ab133989 western blot cocktail.
I hope this has been helpful. Please do not hesitate to let me know if you have any further questions.

Thank you for contacting us at abcam. Please see below under each question the answers.



Questions for ab110170:

1. Step 3 of the protocol states: Resuspend the cells to 5 mg/ml in Reagent A in a 2-ml microtube. What does the 5 mg/ml refer to?

A: 5mg/ml is the protein concentration that customer determined using a protein quantity assay, e.g. BCA assay.

2. Step 6 of the protocol states: Homogenize the cells with 30 strokes using pestle B. What brand of dounce homogenizing system do you use and/or what is the diameter size of pestle B?

A: We get the Dounce homogenizer from VWR https://us.vwr.com/store/catalog/product.jsp?catalog_number=KT885300-0002



i.e. Large pestle (A) clearance = 0.889–0.127 mm; Small pestle (B) clearance = 0.013–0.064 mm



Questions for ab110168:



1. Step 4 of the protocol states: Add up to 2.0 mL of isolation buffer to the tissue in the homogenizer. Is their a general rule to use for volume of isolation buffer per weight of tissue?

A: adding four volumes of buffer to one volume of tissue sample will usually be a good start.



2. Step 5 of the protocol states: To rupture the cells, perform the number of dounce strokes suggested in table 1. Use pestle A for the initial strokes, then use pestle B for the remaining strokes. What brand of dounce homogenizing system do you use and/or what are the diameter size of pestles A and B?



A: same Answer as above.

The size of the pellet after adding reagent B really depends on your starting material which is the number of cells you started your experiment with. It is possible that the pellet is so small that you cannot see it with the naked eye.


In response to your second question, We have tested this kit with BCA assay. But having said that I dont see any issues arising from you using a bradford kit. However we cannot guarantee results will be acheived using the bradford method as we have no data to show it works in any other protein estimation assay apart from BCA.

There may be some minor contamination with other organelles including peroxisomes following use of ab110170 to isolate mitochondria. As you mentioned, it’s very hard to get the mitochondria very clean, and usually requires second round gradient centrifugation.

We would recommend blotting product with mitochondria and peroxisome marker to identify relative levels of organelles in different fractions.
Essentially very pure mitos are best prepared by taking crude mitos and spinning over a gradient (traditionally percoll, metrizamide sometimes used too). References for this technique are given through the links below:
http://books.google.com/books?id=dYKC0YyqKhMC&pg=PA19&lpg=PA19&dq=mitochondria+metrizamide&source=bl&ots=c_V1UkWKxD&sig=p-spEKmCY_2w6jZYhyDsmb02g6s&hl=en&sa=X&ei=-PSSUL6XFYzuigKsloC4DQ&ved=0CEsQ6AEwBA#v=onepage&q=mitochondria%20metrizamide&f=false
2. http://www.ncbi.nlm.nih.gov/pubmed/18600228
In either event this kit could be used. I would recommend using it, if the mitos are pure enough they can be used, if not they can be further purified by gradient centrifugation.

I’d like to emphasize that in the booklet, the expected mitochondrial yield from the example cell lines ARE NOT FROM ONE 150mm plate. If your customer was talking about the yield from only one plate, 200-300mg/plate is very reasonable.



Other than that, the main reason causes low mitochondrial yield is insufficient homogenization. The homogenization step can to be monitored under microscope. A good yield of mitochondria needs about 70% cells to be ruptured after homogenization step. The mitochondrial should be able to pelleted at the recommended speed. (12,000g, 10min) If your customer has concerns about it, it is OK for he/she to do a longer spin, i.e. 12,000g, 20min, 4C.