Mitochondria cytosol fractionation kit

Manufactured by Merck Group

Sourced in United States

The Mitochondria/cytosol fractionation kit is a laboratory tool designed to separate and isolate mitochondria and cytosol fractions from cell samples. It provides a standardized procedure for the efficient extraction and purification of these cellular components.

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Lab products found in correlation

Ab14734, by Abcam (1 mentions) Ab14744, by Abcam (1 mentions) Image studio lite software, by LI COR (1 mentions)

Close spelling variants of this product

ProteoExtract® Cytosol/Mitochondria Fractionation Kit

7 protocols using mitochondria cytosol fractionation kit

Mitochondrial Isolation and Cytochrome C Analysis

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For mitochondria isolation, cells were harvested and then fractionated using a Cytosol/Mitochondria Fractionation Kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer’s recommendations. The cytosol extract was then subjected to Western blotting analysis for Cytochrome C.

Jia G., Wang Q., Wang R., Deng D., Xue L., Shao N., Zhang Y., Xia X., Zhi F, & Yang Y. (2015). Tubeimoside-1 induces glioma apoptosis through regulation of Bax/Bcl-2 and the ROS/Cytochrome C/Caspase-3 pathway. OncoTargets and therapy, 8, 303-311.

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Cytochrome c Translocation Dynamics

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Cells were pretreated with 20 nmol/l of APG-2575 for 0, 0.5, 1, 3, and 6 h. Cytoplasmic fractionation was isolated using the Cytosol/Mitochondria Fractionation kit (#QIA88; Merck Millipore, Darmstadt, Germany). Cytosolic fractions were isolated from OCI-LY8 cells following the manufacturer’s instruction. The amount of cytochrome c in cytosol and mitochondria fraction was determined by Western blot analysis as described above.

Luo Q., Pan W., Zhou S., Wang G., Yi H., Zhang L., Yan X., Yuan L., Liu Z., Wang J., Chen H., Qiu M., Yang D, & Sun J. (2020). A Novel BCL-2 Inhibitor APG-2575 Exerts Synthetic Lethality With BTK or MDM2-p53 Inhibitor in Diffuse Large B-Cell Lymphoma. Oncology Research, 28(4), 331-344.

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Subcellular Fractionation of Tissue

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Dissected hemispheres (prepared as in western blot analysis) were immediately homogenized in 300 μL of ice-cold cytosol extraction buffer (Cytosol/Mitochondria Fractionation kit; Merck, Rockland, Massachusetts, USA) with a protease inhibitor cocktail and DTT. The homogenates were then centrifuged at 700 g for 10 min at 4 °C, and the supernatant was further centrifuged at 10000 g for 30 min at 4 °C. The supernatant obtained after centrifugation at 10000 g was collected as the cytosolic fraction, and the pellet contained the mitochondria. The pellet was resuspended in 50 μL mitochondrial extraction buffer mix containing protease inhibitors and DTT for 10 s and saved as mitochondrial fraction, or maintained intact in PBS at − 80 °C until use.

Wu C.H., Chen C.C., Hung T.H., Chuang Y.C., Chao M., Shyue S.K, & Chen S.F. (2019). Activation of TrkB/Akt signaling by a TrkB receptor agonist improves long-term histological and functional outcomes in experimental intracerebral hemorrhage. Journal of Biomedical Science, 26, 53.

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Mitochondria and Nucleus Fractionation

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A Millipore mitochondria/cytosol fractionation kit was used for extraction of mitochondria and nuclear/cytoplasm extraction reagents were used for separation of nuclei from cytoplasm. For subfractionation of mitochondria, see Supplemental Experimental Procedures.

Liang H., He S., Yang J., Jia X., Wang P., Chen X., Zhang Z., Zou X., McNutt M.A., Shen W.H, & Yin Y. (2014). PTENα is a PTEN isoform Translated through Alternative Initiation and Regulates Mitochondrial Function. Cell metabolism, 19(5), 836-848.

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Mitochondrial Fractionation and Immunoblotting

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Mitochondrial fractions from mock and agonist treated primary neurons were isolated using the mitochondria/cytosol fractionation kit (Millipore). Immunoblotting was done using anti-rabbit SARM1 (Genetex GTX77621) as previously described (19 (link)). Antibodies to voltage-dependent anion channel 1(VDAC1) (Abcam ab14734) or cytochrome c oxidase subunit IV (COXIV) Abcam ab14744) were used as a mitochondrial loading controls. Protein was detected using a Typhoon scanner and analyzed with Image Studio Lite software (LI-COR Biosciences).

Mukherjee P., Winkler C.W., Taylor K.G., Woods T.A., Nair V., Khan B.A, & Peterson K.E. (2015). SARM1, not MyD88, mediates TLR7/TLR9-induced apoptosis in neurons. Journal of immunology (Baltimore, Md. : 1950), 195(10), 4913-4921.

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Cytosol-Mitochondria Fractionation Assay

Cited 2 times

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The cytosol-mitochondria fractionation assay was performed using a Mitochondria/Cytosol Fractionation Kit (Millipore) as previously described^{46 (link), 47 (link)}. Briefly, NHLFs were transfected for 30 h with either non-targeting control siRNA or ISG15-specific siRNA and then transfected with EMCV-RNA or RABV_Le for 16 h. Cells were homogenized in an isotonic buffer using a Dounce homogenizer and the lysates were centrifuged at 600 ×g to pellet the nuclei and unbroken cells. The supernatant was further centrifuged at 10,000 ×g at 4°C for 30 min to separate the cytosolic (supernatant) and mitochondrial (pellet) fractions. The protein concentration of both fractions was determined by a bicinchoninic acid (BCA) assay (Pierce), and equal amounts of proteins were analyzed by immunoblotting. Anti-α-tubulin and anti-MAVS immunoblotting served as markers for the cytosolic and mitochondrial fractions, respectively.

Liu G., Lee J.H., Parker Z.M., Acharya D., Chiang J.J., van Gent M., Riedl W., Davis-Gardner M.E., Wies E., Chiang C, & Gack M.U. (2021). ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity. Nature microbiology, 6(4), 467-478.

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Cytosol-Mitochondria Fractionation for Virus Studies

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The cytosol-mitochondria fractionation assay was performed using a Mitochondria/Cytosol Fractionation Kit (Millipore) as previously described^{48 (link), 49 (link)}. Briefly, NHLFs were transfected for 24 h with either non-targeting control siRNA or ISG15-specific siRNA and then transfected with EMCV-RNA or RABV_Le for 16 h. Cells were homogenized in an isotonic buffer using a Dounce homogenizer and the lysates were centrifuged at 600 ×g to pellet the nuclei and unbroken cells. The supernatant was further centrifuged at 10,000 ×g at 4°C for 30 min to separate the cytosolic (supernatant) and mitochondrial (pellet) fractions. The protein concentration of both fractions was determined by a bicinchoninic acid (BCA) assay (Pierce), and equal amounts of proteins were analyzed by immunoblotting. Anti-α-tubulin and anti-MAVS immunoblotting served as markers for the cytosolic and mitochondrial fractions, respectively.

Liu G., Lee J.H., Parker Z.M., Acharya D., Chiang J.J., van Gent M., Riedl W., Davis-Gardner M.E., Wies E., Chiang C, & Gack M.U. (2020). ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease. bioRxiv.

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