HepaRG cells were plated in 6-well plates and treated as described above. The cells were harvested, lysed with RIPA buffer for 30 min on ice. Lysates were centrifuged at 12,000 rpm for 10 min at 4°C. Total protein concentration was determined using the bicinchoninic acid assay (BCA) protein assay kit. In a parallel experiment, cytosolic and mitochondrial extracts were prepared using the ProteoExtract® Cytosol/Mitochondria Fractionation Kit (Millipore, Billerica, MA, USA) following the manufacturer's protocol. An equal amount of proteins was separated by electrophoresis on SDS-polyacrylamide gels (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with TBST buffer containing 5% skim milk for 1 h, and then incubated with specific primary antibodies overnight at 4°C. After three times washing with TBST buffer, the membranes were further incubated with the horseradish peroxidase-conjugated secondary antibodies (cat. no. 3700; Cell Signaling Technology) at room temperature for 1 h (37 (link),38 (link)). An enhanced ECL detection system was used for visualization of target proteins (iNtRON Biotechnology, Seongnam, Korea).
Proteoextract cytosol mitochondria fractionation kit
Manufactured by Merck Group
Sourced in United States
The ProteoExtract® Cytosol/Mitochondria Fractionation Kit is a laboratory tool designed to separate and isolate cytosolic and mitochondrial fractions from cell samples. It provides a reproducible method for the efficient extraction and enrichment of these cellular components.
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9 protocols using proteoextract cytosol mitochondria fractionation kit
1
Western Blot Analysis of Cellular Fractions
2
Mitochondrial Protein Fractionation
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The mitochondrial and cytosolic KC fractions were extracted using a ProteoExtract® Cytosol/Mitochondria Fractionation Kit (QIA88, Millipore, Calbiochem, USA) as per manufacturers’ instructions. Cytochrome C oxidase subunit 4 (COX4) was used as the control for the mitochondrial fraction. Equivalent protein amounts from the cytosolic and mitochondrial fractions were analysed by western blotting.
3
Rhein-Induced Cytosolic and Mitochondrial Protein Expression in HepaRG Cells
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HepaRG cells were seeded in 6-well plates and incubated with various concentrations of rhein. Then, the cells were collected and lysed with ice-cold RIPA buffer for 30 min. Subsequently, the lysates were centrifuged for 10 min at 12,000 rpm. A bicinchoninic acid assay (BCA) protein assay kit (Dinguo Changsheng Biotechnology, Beijing, China) was used to determine total protein concentration of the supernatant. In a parallel experiment, the mitochondrial and cytosolic fractions were separated using the ProteoExtract® Cytosol/Mitochondria Fractionation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done by loading equal amounts of target protein per lane and then transferring to a polyvinylidene fluoride (PVDF) membrane (Pall, New York, USA). The membranes were blocked with 5% skim milk in TBST (25 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4) buffer for 1 h and then incubated with primary antibodies overnight at 4 °C [54 (link)]. After being washed four times with TBST, the membranes were further incubated with corresponding secondary antibodies at room temperature for 1 h. The target proteins were visualized with an ECL Western blotting detection reagent (Pierce, Appleton, WI, USA). All the experimental results were repeated at least three times.
4
Cytosol/Mitochondria Fractionation and Western Blot Protocol
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RAW 264.7 cells were seeded in 6-well plates and incubated with various concentrations of HB. Then the cells were collected and lysed with ice-cold RIPA buffer for 30 min. Subsequently, the lysates were centrifuged for 10 min at 12,000 rpm. A BCA protein assay kit was used to determine total protein concentration of the supernatant. In a parallel experiment, the mitochondrial and cytosolic fractions were separated by the ProteoExtract® Cytosol/Mitochondria Fractionation Kit (Millipore, Darmstadt, Germany) in accordance with the manufacturer’s instructions. For sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), an equal amount of target protein was loaded per lane and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skim milk in TBST (25 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4) buffer for 1 h, and then incubated with primary antibodies overnight at 4 °C [28 (link)]. After being washed four times with TBST, the membranes were further incubated in corresponding secondary antibodies at room temperature for 1 h. The target proteins were visualized with an ECL western blotting detection reagent (Pierce, Appleton, WI, USA). Semi-quantitation of the scanned films was performed using Quantity One (Bio-Rad, Hercules, CA, USA). All the experimental results were repeated at least thrice.
5
Mitochondrial and Cytosolic Fractionation of HepaRG Cells
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HepaRG cells were handled with polyphyllin VI for 24 h, washed thrice with PBS, and lysed on ice for 30 min using RIPA buffer [150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS]. ProteoExtract® Cytosol/Mitochondria Fractionation Kit (Millipore, Cambridge, MA, USA) was used to isolate the mitochondrial and cytosolic fractions. The lysate was centrifuged at 12,000 rpm for 15 min to remove the insoluble protein. The protein concentrations were measured by BCA protein assay kit. Proteins were electrophoresed using 15% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk and washing with TBST for 1 h, the membranes were incubated with primary antibodies (1:1000) overnight and horseradish peroxidase-conjugated (1:1000) secondary antibodies for 1 h at 4 °C, and then washed thrice in TBST buffer. The target proteins were visualized by an enhanced ECL detection system (iNtRON Biotechnology, Seongnam, Korea).
6
Protein Expression Analysis in Cells
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The treated cells were harvested and lysed with 20% SDS containing 1 mM phenylmethylsulfonyl fluoride. The lysate was sonicated for 1 min on ice followed by centrifugation at 12,000g for 30 min at 4 ºC. Mitochondrial and cytosolic fractions were isolated by using the ProteoExtract® Cytosol/Mitochondria Fractionation Kit (Merck Millipore, Billerica, MA, USA). Then a sample of protein from the supernatant was resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with TBS buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 5% nonfat milk, the membrane was incubated with antibodies against type I procollagen, MMP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies and then was visualized with an ECL chemiluminescence detection kit (PerkinElmer Life Sciences, Waltham, MA, USA). The relative density of the immunoreactive bands was quantified by using a luminescent image analyzer (LSA-100, Fujifilm, Japan).
7
Quantitative Mass Spectrometry of Mito-Esc
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Initially, mitochondrial and cytosolic fractions were separated using a commercially available kit (ProteoExtract Cytosol/Mitochondria Fractionation Kit, Merck, USA) according to manufacturer’s instructions. Mito-Esc was quantified in the mitochondrial and cytosolic fractions obtained from HAEC and aortas of ApoE−/− mice of different treatment groups as mentioned in the Animal Experiments section. Electrospray ionization (ESI)-mass spectrometry (MS) measurements (positive mode) were performed using a quadrupole time-of-flight mass spectrometer (QSTAR XL, Applied Biosystems/MDS Sciex, Foster City, CA). The data acquisition was under the control of Analyst QS software (Applied Biosystems). For the CID (collision-induced dissociation) experiments, the precursor ions were selected using the quadrupole analyzer and the product ions were analyzed using the TOF analyzer47 (link).
The mitochondrial/cytosolic extracts (50 μl) were diluted with 50 μl of methanol and introduced into the ESI source (injection volume 20 μl) using methanol: water (80:20, v/v) as a mobile phase gradient with flow rate 600 μl/min. Stock solution (0.5 mM) of Mito-Esc was made in methanol: water (50:50, v/v). For spiking experiments, appropriate volumes of standard solutions (1–50 μM) were added to the mitochondrial/cytosolic extracts of untreated samples.
The mitochondrial/cytosolic extracts (50 μl) were diluted with 50 μl of methanol and introduced into the ESI source (injection volume 20 μl) using methanol: water (80:20, v/v) as a mobile phase gradient with flow rate 600 μl/min. Stock solution (0.5 mM) of Mito-Esc was made in methanol: water (50:50, v/v). For spiking experiments, appropriate volumes of standard solutions (1–50 μM) were added to the mitochondrial/cytosolic extracts of untreated samples.
8
Mitochondrial Fractionation Using ProteoExtract
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Mitochondrial fractionation of the cells was performed using the ProteoExtract® Cytosol/Mitochondria Fractionation Kit (EMD Millipore), according to manufacturer protocol.
9
Mitocur-1, 2, 3 Fractionation in MCF-7 Cells
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MCF-7 cells were grown in 90-mm dishes, and then treated with or without 10 µM of Mitocur-1, 2, 3 or curcumin for 6 h. After the treatment, cells were washed thrice with PBS. The isolation of mitochondrial and cytosolic extracts was carried out using a commercially available ProteoExtract Cytosol/Mitochondria Fractionation Kit (Merck, USA) according to manufacturer's instructions.
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