Mitochondria Isolation Kit for Cultured Cells (ab110170)

Thank you for your enquiry.

We do not have set expiration dates for our products. We guarantee all of our products to work for at least six month un-opened from the date of purchase when stored correctly.

Please refer to the protocol booklet of the product for specific instruction after opening the single reagents in the kit.


The main difference that MS852 isolates relatively intact mitochondria.
The ab109719 prepares fraction of solubilized mitochondrial proteins, it does not isolate intact mitochondria.
The mitochondrial purity using these two kits is comparable, when started from fresh cultured cells.

Both kits will do a good job to remove cytosolic and nuclear proteins. The “Mitochondrial” fraction of ab109719 is contaminated with proteins of other membrane compartments such as ER, golgi, plasma membrane and vacuoles;
the mitochondria isolated with ab110170 are contaminated also with other membrane compartments that will sediment under 12,000 g, mainly heavy membranes such as ER.
The purity of mitochondrial isolated with ab110170 is very dependent on the user. Also the protocol can be modified to obtain less-contaminated mitochondria. Also mitos prepared by ab110170 can be used as a starting material for further mitochondria purification, e.g. by sucrose gradient centrifugation.


I hope this information helps. Please do not hesitate to contact us if you need anything further.

Thank you for contacting us.

Samples may be prepared as purified mitochondria, crudemitochondria. In most cases whole tissue or cell lysates aresuitable but these may require some sample optimization. Mitochondria samples can be prepared bysimple differential centrifugation of homogenized samples. While any crude isolation may be effective, we recommend ourhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.htmlhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.htmlorhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-with-Dounce-Homogenizer-ab110171.html(with Dounce homogenizer) if using cultured myocytes such as HL-1 cells.If using heart tissues we would recommendhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.htmlhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.htmlorhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-with-Dounce-Homogenizer-ab110169.html(with Dounce homogenizer).



This kit was optimized to extract complex I into its native form (with all 47 subunits assembled in to a large complex). The sample extraction is native so care needs to be taken to extract the samples at equivalent starting concentrations to maintain the protein: detergent ratio and maintain enzyme integrity and activity.Specifically for this product - the extraction is performed by adding 1/10 volume of the 10X Detergent to samples at 5.5mg/mL



I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.




Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Thank you for your reply.

This kit has only been validated for use on cell monolayers and is not optimized for tissue or cells grown on a matrix. The kit yields enriched (not pure) cytosolic, mitochondrial and nuclear fractions, but really what we call “mitochondrial fraction” is a “membrane fraction” (containing mitochondria, plasma membrane, ER, lysozome, etc). If you are looking for ECM proteins that are strongly associated with the plasma membrane, then they will likely be present in the “mitochondrial” fraction. This also means that mitochondrial and ECM proteins will not be separated from each other with this kit.

I hope this helps, please let me know if you need any additional information or assistance.

Thank you for contacting us.

The reason you are not obtaining a pelletis that your starting cell number is too low. In the protocol we suggest for customers to start from 2 to 4 (150mm) plates. Each of these plates has a growth surface of about 175cm2. 1 confluent 150mm dish with C2C12 will hold approximately 1.5x10^7 cells. Which means that you will require at least 3 – 6 x 10^7 cells to generate mitochondria. The kit is not meant to be used for high throughput isolation of mitochondria from cells grown on smaller wells. It is more for isolation from large batches of cells.

If you are unable to generate that number of cells, there is another option. We sell a high throughput cell fractionation kit https://www.abcam.com/Cell-Fractionation-Kit-HT-ab109718.html. It isolates a "mitochondria containing fraction." It is not pure mitochondria, it simply allows youto enrich for mitochondria. With this protocol, you will only generate 50uL of "mitochondria containing fraction."


I hope this helps, please let me know if you need any additional information or assistance.

Thank you for your inquiry.

The protocol you are referring tois not a MitoSciences Protocol that is used in the lab. But it describes a general procedure of mitochondrial isolation.

I can confirm thatNKM buffer is an isosmotic buffer that commonly used to wash cells.

The homogenate buffer (10mMTris,….) is a hypotonic buffer that is used to swell the cells and make them easy to be ruptured.


Sucrose is a compound used to hold an isotonic environment and maintains mitochondria intact.

I could not find the use of Tween 20 or 0.5M KCl in the protocol, therefore, I can not give specific comments on them. In general, Tween 20 is a common detergent while KCl is a chemical compound.


I can recommend our mitochondrial isolation kit (ab110170) for your experiments which is easy anf fast to use.


https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.html (or use the following: https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.html).

I hope this information is helpful and wish you good luck for your experiments.

The laboratory has confirmed that the kit ab110170 can be used to isolate fractions of nuclear and cytosolic proteins in addition to a mitochondria fraction:

"The customer can use ab110170 to isolate nuclear and cytosolic fractions.
The first spin pellet can be considered as enriched nuclear fraction.
The supernatant from the spin of collecting mitochondrial pellet can be considered as cytosolic fraction.
But keep in mind, the nuclear fraction prepared with this kit may still contain intact cells, and the cytosolic fraction may contain other subcellular organelle markers if the homogenate is overdone."

I hope this is clear. Please let me know if you have any additional questions.

We have not tested activity of hexokinase after isolating fromthe mitochondria. But we know using current protocol, we can detect hexokinase on a 2D electrophoresis.


Hope this is helpful. Please feel free to contact us again if you have any further questions.

Thank you for contacting us. I have attached the protocol to this email. For future reference, we do have a protocols tab near the top of each datasheet where you can pull up the kit protocols. The second step of the kit actually calls for you to freeze and thaw the cells to weaken the cell membrane, so yes frozen cells are fine!


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Thank you for your inquiry.

I have to confirm that tissues in 70% Ethanol are not suitable for mitochondrial isolation but can be used for IHC staining instead.

Fresh blood cells are a suitable material to start with. Ifyou areonly interested in the mtDNA, I can recommendthe mitochondrial DNA isolation kit (ab65321).

https://www.abcam.com/Mitochondrial-DNA-Isolation-Kit-ab65321.html (or use the following: https://www.abcam.com/Mitochondrial-DNA-Isolation-Kit-ab65321.html).

If you are also interested in study mitochondrial protein or function,I suggest touse mitochondrial isolation kit (ab110170).

https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.html (or use the following: https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.html).

I confirm that the homogenization step in both protocols will have to be optimizedthough, since different tissues/cells require different strength to brake up the cells. Excessive homogenization will damage the mitochondria and cause the loss of mitochondrial components including mtDNA.

I hope this information is helpful.

Thank you for contacting us and reporting the problems this customer has been having with the Mitochondria Isolation Kit for Cultured Cells kits (ab110170 and ab110171).

As mentioned, I have consulted with the lab in MitoSciences to get their opinion on this case. They have informed me that the problem encountered by your customer is most likely due to the homogenize step being over-done. In order to resolve this, the customer can either add DNAnase (such as benzonase) to clean up the DNA contamination or repeat the mitochondrial isolation with less and gentlerstrokes since her sample was frozen cells.

I hope this information has been of help. It is unfortunate that the customer did not contact us earlier in order for us to be able to help her. I'm glad she is now aware of the Abpomise.

If you have any further questions, please do not hesitate to contact us again.