Mitochondria/Cytosol Fractionation Kit (ab65320)

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

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!

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.

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.

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.

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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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.

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.

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/index.html?datasheet=110169 (or use the following: https://www.abcam.com/index.html?datasheet=110169).

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.