Mitochondria cytosol fractionation kit

To confirm the protein expression of Parkin translocation to the mitochondria, Beta TC-6 mouse pancreas islet cells β cells were harvested in Mitochondria/Cytosol Fractionation Kit (Abcam, ab65320). Briefly, single pancreatic cells (1x10⁷) from which the culture medium was cleared were placed in 1 ml of 1x Cytosol Extraction Buffer Mix (containing DTT and Protease Inhibitors) at 4°C for 10 minutes. Single pancreatic cells were homogenized by passing through a chilled tissue grinder 50 times. At this time, it was checked whether there were any shining cells (cell membranes) from passage 40. Homogenize cells were centrifuged at 600 × g (3000 rpm) to discard the pellet containing the nucleus and damaged cells, and the supernatant was collected. In the supernatant transferred to a new tube, the re-supernatant was collected for the cytoplasmic fraction via a speed centrifuge at 10,000 x g (14,000 rpm) for 30 min. Here, the pellet was solubilized with 100 μl of the Mitochondrial Extraction Buffer Mix (containing DTT and protease inhibitors) for mitochondrial fractionation. VDAC antibody was used as a marker of mitochondria and tubulin antibody was used as a cytoplasmic marker.

PC12 cells and mouse brain tissues were collected and lysed in RIPA buffer (GenDEPOT, Katy, TX, USA) with 1× protease and phosphatase inhibitor cocktail (GenDEPOT) for 30 min and then centrifuged at 12,000 rpm for 30 min at 4 °C.
Mitochondrial and cytosolic proteins in the PC12 cells were assayed with a Mitochondria/Cytosol Fractionation Kit (Abcam). All protein contents were measured by the Bradford protein assay (Bio-Rad).

A Mitochondria/Cytosol Fractionation Kit from Abcam (Cambridge, UK) was used to extract mitochondrial and cytosolic fractions. In brief, 2 × 10⁷ cells were resuspended in 0.5 ml of 1X Cytosol Extraction Buffer Mix containing DTT and protease inhibitors. After incubation on ice for 10 min, cells were homogenized in an ice-cold Dounce tissue grinder (80–100 passes with the grinder). The homogenate was centrifuged at 700×g in a microcentrifuge for 10 min at 4 °C and the supernatant was then centrifuged at 10,000×g in a microcentrifuge for 30 min at 4 °C. After this, the supernatant was collected (cytosolic fraction) and the pellet (intact mitochondria) was re-suspended in 50 μl of the Mitochondrial Extraction Buffer Mix containing DTT and protease inhibitors (mitochondrial fraction). For mitochondrial reactive oxygen species (mtROS) measurement, cells were incubated with 5 μM MitoSOX™ red (Invitrogen) in culture medium for 30 min at 37 °C. After washing with PBS, the cells were detached by trypsin treatment and analyzed with flow cytometry [28 ].

The mitochondrial and cytosolic proteins in C28/I2 cells were isolated with the Mitochondria/Cytosol Fractionation Kit (Abcam) and were supplemented with a protease inhibitor cocktail (Roche). Then the protein samples were separated with SDS/PAGE (12% gel) and were electrotransferred on to nitrocellulose membranes (Millipore). The membranes were incubated by rabbit antibody against Cyt c, CASP 3, CREB, CREB with Ser¹³³ phosphorylation, RhoA (all Santa Cruz Biotechnology) or β-actin (Sigma–Aldrich). Then, the specific binding signal was acquired using the ECL detection systems with the incubation with horseradish-peroxidase–conjugated secondary antibodies (Pierce).

Cell fractionation was performed using the Mitochondria/Cytosol Fractionation Kit (ABCAM, Cambridge, U.K.). After seeding 10 million cells and treating them as reported above, the medium was removed, and the cells were collected in 15 mL tubes and centrifuged at 600× g for 5 min at 4 °C. Subsequently, the supernatant was removed, and the pellet was resuspended in 5 mL of cold PBS. It was centrifuged at 600× g for 5 min at 4 °C. The supernatant was removed, and the pellet was resuspended with 1 mL of Cytosol Extraction Buffer Mix 1×. It was incubated on ice for 10 min, and then the cells were homogenized with the Potter. The homogenate was transferred to 1.5 mL tubes and centrifuged at 700× g for 10 min at 4 °C. The supernatant was collected in other 1.5 mL tubes and centrifuged at 10,000× g for 30 min at 4 °C. The supernatant, i.e., the cytosolic fraction, was taken and placed in other tubes, while the pellet, i.e., the mitochondrial fraction, was resuspended with YY lysis buffer, and the samples were prepared for the electrophoretic run. In the case of cell fractionation, COX4, a mitochondrial protein, and PP2A, a protein localized in the cytosol, were used as control proteins. The detection of these proteins in the cytosolic and mitochondrial fractions provides control of the purity of the obtained fractions.