Mitochondria were isolated using Biovision Mitochondria/Cytosol Fractionation Kit (Biovision, K256). Briefly, 5 × 107 cells were collected and homogenized using an ice-cold douncer in mitochondrial isolation buffer containing DTT and protease inhibitors. After 40 strokes, samples were centrifuged at 700 g for 10 min at 4°C and the supernatant were centrifuged at 10,000 g for 30 min at 4°C to obtain the mitochondrial fraction. Pellets were resuspended in 100 μl 1x PBS for protein quantification. 50 μg of mitochondrial fraction was prepared bya centrifugation at10,000 g for 10min at4°C,and resuspended in 20 μl NativePAGE samplebuffer containing 5% digitonin (Invitrogen). BN-PAGE was performed as previously described (Jha et al., 2016 (link)). To detect CIII and CIV of the electron transport chain, antibodies were selected based on the amino acid sequence-identity of the used immunogens to generate the antibodies. Anti-UQCRC2 (1:1,000, ab14745, Abcam) and anti-MTCO1 (1:1,000, ab14705, Abcam) were used. After incubation with the primary antibody dilution, the membrane was washed and developed using the WesternBreeze Chromogenic Western Blot Immunodetection Kit (Invitrogen).
Biovision mitochondria cytosol fractionation kit
Manufactured by Abcam
Sourced in United States
The Biovision Mitochondria/Cytosol Fractionation Kit is a laboratory tool used for the separation and isolation of mitochondria and cytosol from cultured cells or tissue samples. The kit provides a simple and efficient method to obtain purified mitochondrial and cytosolic fractions for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Digitonin, by Thermo Fisher Scientific (2 mentions)
Ab14745, by Abcam (2 mentions)
Ab14705, by Abcam (2 mentions)
Westernbreeze chromogenic western blot immunodetection kit, by Thermo Fisher Scientific (2 mentions)
Anti acetylated α tubulin, by Merck Group (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti bax, by BD (1 mentions)
4 protocols using biovision mitochondria cytosol fractionation kit
1
Mitochondrial Fractionation and BN-PAGE
2
Fibroblast Mitochondrial Fractionation and Western Blotting
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Primary human skin fibroblasts were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and protease inhibitors) and subjected to western blotting as previously described [23 (link)]. For some experiments, we separated the mitochondrial and cytosolic fractions from fibroblast lysates using the BioVision Mitochondria/Cytosol Fractionation kit (BioVision, Milpitas, CA, USA). The following primary antibodies were used: anti-acetylated α-tubulin (1:1000; Sigma-Aldrich), anti-tyrosinated α-tubulin (1:1000; Millipore), anti-α-tubulin (1:1000; Sigma-Aldrich), anti-BAX (1:1000; BD, Franklin Lakes, NJ, USA), and anti-GAPDH (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA).
3
Rapid Mitochondrial Fractionation from Cells
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Cell lysates were separated into cytosolic or mitochondrial fractions using the BioVision Mitochondria/Cytosol Fractionation Kit (BioVision #K256) according to the manufacturer’s protocol. Briefly, cells were lysed by passing cells through a 25 g needle 25–30 times. Unbroken cells and nuclei were cleared by centrifugation at 700 g until no pellet was observed. The samples were then spun at 10,000 g and the supernatant was saved as the cytosolic fraction. The remaining pellet was resuspended in mitochondria extraction buffer as the mitochondrial fraction.
4
Mitochondrial Fractionation and BN-PAGE
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Mitochondria were isolated using Biovision Mitochondria/Cytosol Fractionation Kit (Biovision, K256). Briefly, 5 × 107 cells were collected and homogenized using an ice-cold douncer in mitochondrial isolation buffer containing DTT and protease inhibitors. After 40 strokes, samples were centrifuged at 700 g for 10 min at 4°C and the supernatant were centrifuged at 10,000 g for 30 min at 4°C to obtain the mitochondrial fraction. Pellets were resuspended in 100 μl 1x PBS for protein quantification. 50 μg of mitochondrial fraction was prepared bya centrifugation at10,000 g for 10min at4°C,and resuspended in 20 μl NativePAGE samplebuffer containing 5% digitonin (Invitrogen). BN-PAGE was performed as previously described (Jha et al., 2016 (link)). To detect CIII and CIV of the electron transport chain, antibodies were selected based on the amino acid sequence-identity of the used immunogens to generate the antibodies. Anti-UQCRC2 (1:1,000, ab14745, Abcam) and anti-MTCO1 (1:1,000, ab14705, Abcam) were used. After incubation with the primary antibody dilution, the membrane was washed and developed using the WesternBreeze Chromogenic Western Blot Immunodetection Kit (Invitrogen).
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