Mitochondria cytosol fraction kit

Total protein was extracted from cell samples by using RIPA buffer (Beyotime). 50 μg of the extracted and the co-precipitated proteins were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking in 5% nonfat milk for 2 h at room temperature, the membranes were incubated with anti-p-Bad (1:200, Santa Cruz), anti-p-AKT (1:200, Santa Cruz), anti-PTEN (1:200, Santa Cruz), anti-Bad (1:200, Santa Cruz), anti-AKT (1:200, Santa Cruz), anti-Bcl-2 (1:200, Santa Cruz), anti-cytochrome c (1:200, Santa Cruz), anti-caspase-9 (1:200, Santa Cruz) and anti-caspase-3 (1:200, Santa Cruz). After incubation with primary antibodies, membranes were washed and incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz) for 2 h at room temperature. Proteins on the membrane were visualized by using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA). Glyceraldehyde-3-phosphate (GAPDH) was used as an internal control to ensure equal protein loading. Mitochondria/Cytosol Fraction Kit (BioVision, Milpitas, CA, USA) was used to separate the mitochondria fraction and cytosol fraction before detection of cytochrome c.

For detection of cytochrome c (cyto c) in cytoplasm, mitochondria were removed and the cytoplasm was collected by using Mitochondria/Cytosol Fraction Kit (BioVision, USA). For detection of other proteins, whole cell lysates were prepared by using RIPA lysis buffer. Subsequently, protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, USA). Proteins on the membrane were then incubated with the primary antibodies and probed by appropriate secondary antibodies. Protein bands were finally detected by using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Inc, USA).

Cytosolic and mitochondrial fractions were isolated from cells by a mitochondria/cytosol fraction kit (Biovision, USA) according to the manufacturer’s protocols. The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and mitochondrial inner membrane protein TIM23 were served as standards for cytosol and mitochondrial protein, respectively [92 (link)].

To separate mitochondria from cytoplasm in Huh7 and HepG2 cells, mitochondria/Cytosol Fraction Kit (BioVision, USA) was used according to the manufacturer's protocol. Mitochondria -derived proteins or total proteins in Huh7 and HepG2 were extracted by using radio immunoprecipitation assay (RIPA) lysis buffer (Cell Signaling, USA). Concentrations of each protein sample were determined by BCA Assay Kit (Pierce, USA). Equal amounts of proteins in each sample were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were then probed with primary antibodies (anti-Bcl-2, anti-Mcl-1, anti-Bcl-xl, anti-Bax, anti-Bid, anti-c-FLIP, anti-DR5, anti-DR4, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved PARP, anti-Cox IV and anti-β-actin, Cell Signaling) overnight. Subsequently, the Membranes were incubated with suitable horseradish peroxidase-conjugated secondary antibodies for 2 h. Blots were visualized by using enhanced chemilu-minescence detection kit (Pierce).

To evaluate the protein level of cytochrome c in cytoplasm and mitochondria of Hep-2-CSCs, the mitochondria in cells were isolated using Mitochondria/Cytosol Fraction Kit (BioVision, USA) according to the manufacturer's guidance. Subsequently, western blot analysis was performed to detect the released cytochrome c.

To obtain the protein of Smac/DIABLO in cytoplasm, mitochondria of BCSCs were removed by using Mitochondria/Cytosol Fraction Kit (BioVision, USA) according to the manufacturer’s instructions.