Mitochondria/Cytosol Fractionation Kit

Applikation: Immunpräzipitation (IP)

Produktnummer: ABIN412483

Produktbeschreibung

Beschreibung: The 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.

Anwendungen

Applikationshinweise: Note: To check the efficiency of homogenization, pipette 2-3 μ l of the homogenized suspension onto a coverslip and observe under a microscope. A shiny ring around the cells indicates that cells are still intact. If 70-80% of the cells do not have the shiny ring, proceed to step 7. Otherwise, perform 10-20 additional passes using the dounce tissue grinder. Excessive homogenization should also be avoided, as it can cause damage to the mitochondrial membrane which triggers release of mitochondrial components. 7. Transfer homogenate to a 1.5-ml microcentrifuge tube, and centrifuge at 700 x g (~3000 rpm) in a microcentrifuge for 10 minutes at 4 o C. Collect the supernatant carefully and discard the pellet. 8. Transfer the supernatant to a fresh 1.5-ml tube, and centrifuge at 10,000 x g (~13000 rpm) in a microcentrifuge for 30 minutes at 4 o C. Collect Supernatant and save the pellet. 9. Collect the supernatant from Step 8 as Cytosolic Fraction (Store at –80 o C). 10. If intact mitochondria are desired, resuspend the pellet from Step 8 in 0.1 ml 1X PBS (Not provided). These are the intact mitochondria. If mitochondrial protein lysate is desired, resuspend the pellet from Step 8 with 100 μ l of the Mitochondrial Extraction Buffer Mix containing DTT and protease inhibitors (as prepared in Section A), vertex for 10 seconds and save as Mitochondrial Fraction (Store at –80 o C).

Beschränkungen: Nur für Forschungszwecke einsetzbar

Publikationen

Publikationen: Li, Han, Liu et al.: "Single-prolonged stress induced mitochondrial-dependent apoptosis in hippocampus in the rat model of post-traumatic stress disorder." in: Journal of chemical neuroanatomy, 2010 (PubMed).