Mitochondria/Cytosol Fractionation Kit (ab65320)

Submit a review Q&A (16)References (29)

Key features and details

  • Assay type: Direct
  • Assay time: 4 hr
  • Sample type: Adherent cells, Suspension cells, Tissue

Overview

  • Product name

    Mitochondria/Cytosol Fractionation Kit

  • Sample type

    Tissue, Adherent cells, Suspension cells

  • Assay type

    Direct

  • Assay time

    4h 00m

  • Product overview

    Abcam's Mitochondria/Cytosol Fractionation Kit provides unique formulations of reagents for effective isolation of a highly enriched mitochondrial fraction from cytosolic fraction of mammalian cells including both apoptotic and nonapoptotic cells. The enriched mitochondrial and cytosolic fractions can be used for studying apoptotic and signal transduction pathways to detect translocation of factors interested between the two fractions by Western blotting, ELISA, or other assays. Procedures are simple and easy to perform, no ultracentrifugations and toxic chemicals are involved.
    Visit our FAQs page for tips and troubleshooting.

  • Notes

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

Properties

References (29)

Publishing research using ab65320? Please let us know so that we can cite the reference in this datasheet.

ab65320 has been referenced in 29 publications.

  • Ishikawa T  et al. Protein kinase C promotes choline transporter-like protein 1 function via improved cell surface expression in immortalized human hepatic cells. Mol Med Rep 21:777-785 (2020). PubMed: 31974614
  • Chang CY  et al. Aspirin Induced Glioma Apoptosis through Noxa Upregulation. Int J Mol Sci 21:N/A (2020). PubMed: 32545774
  • Tadokoro T  et al. Mitochondria-dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity. JCI Insight 5:N/A (2020). PubMed: 32376803
  • Ni S  et al. Mitochondrion-mediated iron accumulation promotes carcinogenesis and Warburg effect through reactive oxygen species in osteosarcoma. Cancer Cell Int 20:399 (2020). PubMed: 32831652
  • Roy S  et al. MDM2 Antagonists Induce a Paradoxical Activation of Erk1/2 through a P53-Dependent Mechanism in Dedifferentiated Liposarcomas: Implications for Combinatorial Strategies. Cancers (Basel) 12:N/A (2020). PubMed: 32806555
View all Publications for this product

Customer reviews and Q&As

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1-10 of 16 Abreviews or Q&A

Question



Inquiry: Hello, I would like to know if I can store the cytosolic and mitochondrial buffers for months in the freezer (-20 C) either as a whole or in aliquots and if not then why please. Thanks

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Answer

Thank you for contacting us.



We do not recommend freezing the buffers after first thaw for best results. Some components can precipitate out upon freezing.


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Question



Thank you for your reply. I'm interested in the kits which can isolate mitchondria. So Cell Fractionation Kit Standard (ab109719), Mitochondria/Cytosol Fractionation Kit (ab65320), Mitochondria Isolation Kit for Cultured Cells (ab110170) might fit my request. However, as you see, my focus is the mRNAs attached on mitochondria outer membrane, I'm not sure whether these kits can work.

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Answer

ab109719 is not suitable for this purpose, this kit is based on detergent fractionation of cellular proteins into three fractions. RNA distribution was not tested with this kit.



Mitochondrial isolation kits (ab110168 – ab110171) are designed to isolate whole mitochondrial organelle, as such these kits are suitable to isolate mRNA that is attached to/ within this compartment. I would recommend to supplement all the buffers of the Mitochondrial Isolation Kit with RNAse inhibitors. We do not have any data regarding the yield of mRNA. The reference below maybe useful reading:



http://www.ncbi.nlm.nih.gov/pubmed/6197615

Attardi G, Montoya J.

Methods Enzymol. 1983;97:435-69. No abstract available.

PMID:

6197615



Please let me know if you have any further questions!

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Question

Inquiry: According to the Protocol Booklet [https://www.abcam.com/ps/products/65/ab65320/documents/ab65320%20Mitochondria%20Cytosol%20Fractionation%20Kit%20%28Website%29.pdf], this product is to be used with cells. On the other hand, on the website [https://www.abcam.com/mitochondriacytosol-fractionation-kit-ab65320.html] under "Sample type" you also mention "Tissue". However, there are no specific details on how to use this kit with (frozen) tissue. I've been going through your Q&A section and found a couple of relevant questions, but I'm still pretty puzzled. A major issue is homogenisation. I have never used a Dounce homogeniser/tissue grinder before and I'm not even familiar with it. 1) Is it possible to homogenise the tissue with other tool(s)/ways? 2) If not, how does the Dounce homogeniser/tissue grinder really work? Can it really take a tissue sample? I would appreciate specific answers and instructions please, instead of standard replies. Looking forward to hearing from you.

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Answer


I have been in contact with the lab who have confirmed that fresh tissue can be used with this kit. For this sample type, you need to isolate out the cells from the tissues by enzymatic breakdown and follow the protocol mentioned on the datasheet exactly.

Unfortunately, I suspect that frozen tissues are not going to work with this kit.

As for homogenisation, we have only used a Dounce homogenizer with this kit and therefore we strongly recommend using this over other methods such as using a polytron. Excessive homogenization should be avoided, as it can cause damage to the mitochondrial membrane which triggers release of mitochondrial components. We recommend for each 10 mg of tissue, to use 1 ml of the 1X Cytosolic Extraction Buffer containing DTT and Protease Inhibitors for homogenization. Follow the protocol exactly from step 5 of the assay protocol.

A Dounce homogenizer is an apparatus consisting of a glass tube with a tight-fitting glass pestle used manually to disrupt tissue suspensions to obtain single cells or subcellular fractions.

For more information, see: http://opsdiagnostics.com/notes/ranpri/Homogenization%20Guide%20ver.1.pdf

If you do not have a Dounce homogeniser, may I recommend Mitochondria Isolation Kit for Tissue (with Dounce Homogenizer) (ab110169) which might be more suited to your needs and is specific for tissue samples. In addition, although fresh tissue is recommended to use with this kit, frozen tissue usually gives OK results although some losses may be observed due to the freezing process.

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Question

Thank you for your detailed information:
According to your reply "The supernatant after centrifugation will be mostly cell debris including parts of the membrane. "
Another protein of interest for us is " EP4 receptor" ab45295" what is the recommended centrifugation? I don't think the kit " a b65320" would fit, is that right?

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Answer

Thank you for your reply.

You would probably be best suited using a kit that is able to isolate the membrane fraction completely if you are looking at a membrane protein like Prostaglandin E Receptor EP4. You may want to use a kit like our ab139409, membrane fractionation kit. https://www.abcam.com/membrane-fractionation-kit-ab139409.html

I hope this information helps. Please contact us with any other questions.

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Question

We are interested to purchase the ab65320 from your company. There are couple questions I have regarding the protocol:
Just a little background before start of my question: Our lab research focus is primarily on human skeletal muscle biopsy samples (not cell culture or animal related)
1,Could I directly homogenize sample then centrifuge at 600g or 700g and discard the supernatant and what component are in this supernatant?
2, We would like to know the general chemical content used in your buffer solution
3, Do you have any remomended product can further seperate microsomal protein fractionation?

Look forward to hearing back from you.

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Answer

Thank you for contacting us.

The supernatant after centrifugation will be mostly cell debris including parts of the membrane. This kit is sourced from an outside lab and they consider the components of the buffers to be proprietary so we do not know what is in the buffers. We do not have a specific protocol or kit for microsomal protein fractionation for use with this ab65320, but I did find these protocols online which may be helpful:

http://panomics.pnnl.gov/training/protocols/Cellular_proteinfractionation_ncrr.pdf

http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.1963.tb05057.x/abstract

We do have an ER fraction WB cocktail that will detect ER in fractions as ab139415. https://www.abcam.com/endoplasmic-reticulum-fraction-western-blot-cocktail-ab139415.html

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

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Question

I would like to use this kit for mouse brain tissue.  The instructions are for cells.  Can you tell me how to modify the assay protocol for brain tissue?  For example, how much cytosolic buffer per brain weight, as well as mitochondria extraction buffer?  Do the spin speeds and times need to modified?  

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Answer

Thank you for contacting us.

I have been in contact with the lab regarding your inquiry. For each 10 mg of tissue, use 1 ml of the 1X Cytosolic Extraction Buffer containing DTT and Protease Inhibitors for homogenization. Follow the protocol exactly from step 5 as shown in the attached datasheet.

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

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Question

Dear technical team,

Our customer would like to know if these kits are available to mouse sample.
Please let me know.

Best regards

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Answer

Thank you for your enquiry and your interest in our products.

I can confirm that both kits can be used for mammalian cells (including mouse samples).

If you need any further assistance in the future, please do not hesitate to contact me.

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Question

Inquiry: Dear, I'm writing to you to have an information. I would like order a kit for cytosol and Mitocondrial extraction. I saw your kit: ab109719 and ab65320. Could you, please, explain to me the differences between the kits. I have to extract the fractions from a few number of cells (samples: mouse dendritic cells, 2X106 cells). And I would like measure in the cytosol fraction the Citochrome c release after apoptosis signal. Thank you very much Best Regards

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Answer

Thank you for your enquiry and interest in our products.

https://www.abcam.com/ab109719 - Cell Fractionation Kit Standard: allows for the rapid and simple preparation of mitochondrial, cytoplasmic and nuclear fractions from cultured cells. Unlike competing products, this kit does not require mechanical disruption of the sample. Please take a look at the Protocol booklet for further details:

https://www.abcam.com/ps/products/109/ab109719/documents/ab109719%20cell%20fraction%20standard%20(Website).pdf

https://www.abcam.com/ab65320 - Mitochondria/Cytosol Fractionation Kit: provides unique formulations of reagents for effective isolation of a highly enriched mitochondrial fraction from cytosolic fraction of mammalian cells including both apoptotic and nonapoptotic cells. Procedures are simple and easy to perform, no ultracentrifugations and toxic chemicals are involved. Please take a look at the Protocol booklet for further details:

https://www.abcam.com/ps/products/65/ab65320/documents/ab65320%20Mitochondria%20Cytosol%20Fractionation%20Kit%20(Website).pdf

If you need any further assistance in the future, please do not hesitate to contact me.

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Question

We recently purchased Mitochondria/CytosolFractionation Kit(ab65320) from Abcam. we do not haveDounce tissue grinder, Can we use sonicator toHomogenize cells after addition of1X Cytosol Extraction Buffer Mix.

please suggest what can i do .

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Answer


Please do not use a sonicatorin order to destroy your cells, as this will result in a complete lysis of the mitochondria as well. This means you will not be able to analyse the sample.



However, we do have a kit in which we delivers the grinder as well:

https://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-with-Dounce-Homogenizer-ab110169.html (or use the following: https://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-with-Dounce-Homogenizer-ab110169.html).


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Question

Hallo, I am considering purchasing Ab65320, however I have a couple of questions: my aim is to run WB on total cytoplasmic extract as well as mitochondria free cytoplasmic extract on primary macrophages. I would like to know whether a polytron can replace the Dounce Tissue Grinder in order to homogenize the cells? Also, the cytosolic fraction obtained following your protocol is already mitochondria free, correct? Alternatively, does your company provide a kit that allows me to collect total cytoplasm first and then mitochondria-free cytoplasm on the same sample, or one has to perform lysis on two different samples?

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Answer


Unfortunately we do not have any data regarding the use of the kit ab65320with samples homogenised with a polytron. We recommend the use of a Dounce homogenizer. Excessive homogenization should be avoided, as it can cause damage to the mitochondrial membrane which triggers release of mitochondrial components.

Yes, the cytosolic fraction obtained using ab65320is mitochondria free.

An alternative fractionation kit that may be of interest for you is the Cell Fractionation Kit référence https://www.abcam.com/ab109719. This kit provides a method and reagents for a rapid preparation of cytosolic, mitochondrial and nuclear fractions.https://www.abcam.com/ab109719 is based on sequential detergent-extraction of cytosolic and mitochondrial proteins without the need for mechanical disruption of cells, and thus fractionates cells into cytosol-containing, mitochondria-containing and nuclei-containing fractions.

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