After exposure to 100 or 1000 pg/mL of TNF-α for 3, 6, 12, and 24 h, HT-22 cells were fractioned into cytosolic and mitochondrial fractions using the Mitochondrial/Cytosol Fractionation Kit (Biovision, Milpitas, CA, USA). The protein concentration of each fraction was determined using the Pierce 660 nm protein assay (Thermo Scientific, Waltham, MA, USA). Cytochrome c levels in the cytosolic and mitochondrial fractions were determined using a rat/mouse cytochrome c Quantikine ELISA (R&D Systems, Minneapolis, MN, USA). Percentage release of cytochrome c was calculated using the following equation: total cytosolic cytochrome c/(cytosolic + mitochondrial cytochrome c) (Tanaka et al. 2004 (link)).
Mitochondrial cytosol fractionation kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Mitochondrial/Cytosol Fractionation Kit is a laboratory tool designed to isolate and separate mitochondria and cytosolic fractions from mammalian cells. It provides a simple and efficient method for the preparation of these cellular components for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce 660 nm protein assay, by Thermo Fisher Scientific (1 mentions)
Superoxide dismutase sod activity assay kit, by Abcam (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp labeled anti mouse and anti rabbit secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
Polyclonal bak, by Santa Cruz Biotechnology (1 mentions)
Taq pcr mastermix, by Tiangen Biotech (1 mentions)
Rna extraction kit, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Abcam (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Gemcitabine gemzar, by Eli Lilly (1 mentions)
Superoxide dismutase activity assay kit, by Abcam (1 mentions)
Mr 96a, by Mindray (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab78547, by Abcam (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Immobilon nc, by Merck Group (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab8295, by Abcam (1 mentions)
14 protocols using mitochondrial cytosol fractionation kit
1
Quantifying Cytochrome c Release in HT-22 Cells
2
Measuring Mitochondrial and Cytosolic SOD Activity
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To measure SOD activity from mitochondria and cytosol fractions separately, the Mitochondrial/Cytosol Fractionation Kit from BioVision (Catalog number: K256-25) was used to separate both hepatic fractions, and the Superoxide Dismutase (SOD) Activity Assay Kit from BioVision (100 reactions and a catalog number of K335-100) was then used to measure this enzyme activity (in U/mL) separately.
3
Mitochondrial and Cytosolic Fractionation
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Mitochondrial and cytosolic fractions of cells were prepared using a mitochondrial/cytosol fractionation kit (Biovision). Cells were harvested by centrifugation at 600 × g for 5 min and washed twice with cold PBS buffer. The cells were re-suspended in 250 μL of an extraction buffer, which contained the protease inhibitor mixture and dithiothreitol (Biovision). After incubation on ice for 30 min, the cells were homogenized on ice and centrifuged at 700 × g for 10 min at 4 °C, and the supernatant was collected. The collected supernatant was centrifuged again at 10 000 × g for 30 min at 4 °C. The resulting supernatant was harvested and used as cytosolic fractions. The pellets were re-suspended and used as mitochondrial fractions.
4
Cerebellum Fractionation for Bid and tBid
5
Antibody-Based Protein Expression Analysis
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The following antibodies were used: the primary antibodies for cleaved PARP-1 (cleaved p25), DR5, and Smac/DIABLO were purchased from GeneTex whereas AIF was purchased from Thermo Scientific. Antibodies against antibody against polyclonal caspase-3, monoclonal DR4, monoclonal p21, polyclonal p27, monoclonal Cdk2, polyclonal Cdk4, polyclonal cyclin D1, polyclonal cyclin E, polyclonal cIAP-1, polyclonal c-FLIPL, monoclonal survivin, polyclonal Bak, polyclonal Bax, monoclonal Bcl-xL, polyclonal Bid, polyclonal GADD153, monoclonal p-ERK, polyclonal ERK2 and polyclonal Akt1 were purchased from Santa Cruz. Antibodies against monoclonal caspase-8, monoclonal cytochrome c, monoclonal COX IV, polyclonal p-Rb, monoclonal Rb, polyclonal XIAP, monoclonal p-p38, polyclonal p38, polyclonal p-JNK (Thr183/Tyr185), monoclonal polyclonal JNK, p-Akt (Ser473) and β-actin were purchased from Cell Signaling Technology. The horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit secondary antibodies were purchased from Santa Cruz. Bradford reagent was purchased from Bio-rad. Mitochondrial/cytosol fractionation kit was purchased from BioVision.
6
Mitochondrial and Cytosolic Fractionation
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The mitochondrial and cytoplasmic fractions were separated using the Mitochondrial/Cytosol fractionation kit (BioVision, CA, USA) as per the manufacturer’s instructions. Briefly, whole-cell pellets dissolved in cytosolic fraction extraction buffer were subjected to 55 strokes in a 2 ml Dounce homogenizer on ice. The homogenate was centrifuged at 3,500 rpm for 10 min at 4°C to pellet nuclei and unbroken cells. The supernatant was subsequently centrifuged at 13,000 rpm for 30 min at 4°C to obtain cytosolic supernatant and the mitochondrial pellet. Mitochondrial pellets were resuspended in mitochondrial extraction buffer by gentle vortex for 30 sec.
7
Oridonin Potentiates Gemcitabine-Induced Apoptosis in Pancreatic Cancer
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Oridonin was obtained from the Beijing Institute of Biological Products (Beijing, China). The purity of oridonin was measured by HPLC and determined to be 99.4%. Oridonin was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution at 10 mmol/L concentration and stored at −20 °C. The DMSO concentration was kept below 0.1% in all cell culture experiments and did not exert any detectable effects on cell growth or cell death. Gemcitabine (Gemzar; Eli Lilly and Company) was stored at 4 °C and dissolved in sterile PBS at 0.2 mmol/L concentration on the day of use. The Cell Counting Kit-8 (CCK-8) was obtained from Abcam. Annexin V-FITC/PI apoptosis detection kit was purchased from Biological Development Co. Ltd. The RNA extraction kit was purchased from Life Technologies. cDNA first-strand synthesis kit was obtained from Fermentas. The 2×Taq PCR MasterMix was purchased from TIANGEN and the mitochondrial/cytosol fractionation kit was purchased from BioVision, CA, USA. Fetal bovine serum (FBS), trypsin containing EDTA, Roswell Park Memorial Institute-1640 (RPMI-1640), Ribonuclease A (RNase A), propidium iodide (PI), DMSO, antibodies against Bax, Smac, X-linked inhibitor of apoptosis protein (XIAP), caspase-9, cleaved caspase-3, β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-rabbit and goat-anti-mouse) were all purchased from Sigma.
8
Quantifying Mitochondrial and Cytosolic SOD
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The mitochondrial and cytosol SOD fractions were extracted using the Mitochondrial/Cytosol Fractionation Kit from BioVision (Catalog number: K256-25). The absolute activity (in U/ml) of SOD was estimated using the Superoxide Dismutase Activity Assay kit (Biovision, USA, 100 Rnx, catalog number K335-100) for a colorimetric reading at a wavelength of 450 nm using a microplate Elisa reader (Mindray MR-96A) according to the specified instructions provided by the seller.
9
Isolation of Liver Mitochondria
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Liver mitochondria were freshly isolated by differential centrifugation followed by rapid centrifugation through Percoll density gradient as previously described [20] (link). Alternatively, mitochondria from frozen livers were obtained using the Mitochondrial/Cytosol Fractionation Kit, Abcam (London, UK).
10
Quantification of Cytochrome c in Subcellular Fractions
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For the quantification of cytochrome c, cytoplasm and mitochondria were separated using a Mitochondrial/Cytosol Fractionation Kit (ab65320, Abcam, Cambridge, United Kingdom). Afterward, the cytochrome c content in the mitochondrial and cytosolic fraction was quantified by western blotting using Anti-cytochrome C antibody (ab133504, Abcam, Cambridge, United Kingdom). The purity of the fractions was checked by the determination of TOMM20 (a protein of the outer mitochondrial membrane) and α-tubulin (a major constituent of microtubules in the cytoplasm) by western blotting (Supplementary Figure S1 ). The antibodies used for western blotting were ab78547 (Abcam, Cambridge, United Kingdom) for TOMM20 and ab76290 (Abcam, Cambridge, United Kingdom) for alpha-tubulin. Western blots were performed as described in the following section.
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