Subcellular fractionation was done using a digitonin-based method as described in (Adrain et al., 2001 (link)). Briefly, cells were harvested and centrifuged at 300g for 10min, washed in TBS 2.5mM pH 7.5, and repelleted. Cells were permeabilized for 5min on ice with cytosolic extraction buffer (CEB) (250 mM sucrose, 70 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.2, 1× complete protease inhibitor cocktail, Roche) containing freshly prepared digitonin (200 µg/ml, D-5628, Sigma-Aldrich). Cytosolic fraction was isolated by collecting the supernatant after centrifugation at 1000 g for 5 min at 4 °C. The mitochondrial fraction was washed in CEB buffer with digitonin, CEB buffer and twice with PBS and resuspended in whole cell extraction buffer (WCE) (25mM Hepes pH 7.7, 0.3M NaCl, 1.5mM Mg Cl22, 0.2mM EDTA, 0.1% Triton X-100, 100µg/ml PMSF, 1× complete protease inhibitor cocktail, Roche).
D5628
Manufactured by Merck Group
D5628 is a laboratory equipment product from Merck Group. It is designed for specific laboratory functions. The core function of this product is to perform precise measurements and analysis in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ab110325, by Abcam (1 mentions)
Ab150105, by Abcam (1 mentions)
S3023, by Agilent Technologies (1 mentions)
Mitotracker red cmxros, by Solarbio (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Anti ndufa9, by Abcam (1 mentions)
Mini trans blot cell system, by Bio-Rad (1 mentions)
Immobilon fl 0.45 μm, by Merck Group (1 mentions)
Anti sdha, by Thermo Fisher Scientific (1 mentions)
Anti coi, by Thermo Fisher Scientific (1 mentions)
Sea blocking buffer, by Thermo Fisher Scientific (1 mentions)
3 protocols using d5628
1
Subcellular Fractionation Using Digitonin
2
Visualizing Mitochondrial Cytochrome C Expression
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To detect the expression of mitochondrial cytochrome c, a digitonin-based permeabilization method was employed in this study [52 (link), 53 (link)]. First, 3000 MDA-MB-231, MCF-7 and E0771 cells were seeded on a slide and cultured in a 24-well plate overnight. The cells were treated with 50 ng/mL FGF21 and DOX (1.25 μM for MDA-MB-231 and E0771, 5 μM for MCF-7). The treatment duration was 20 h. After the treatment, the cells were stained with Mitotracker® red CMXRos (M9940, Solarbio) for 45 min. Following staining, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at 37 °C. To permeabilize the cells, a purified digitonin (0.004%, D5628, Sigma) was used for 2 min at room temperature. The digitonin was prepared according to the manufacturer’s protocol.
Next, the cells were incubated with blocking buffer (3% BSA, 0.2% sodium azide, and 0.1% tween-20 in PBS) for 30 min at room temperature. Subsequently, the cells were incubated with primary antibody (ab110325, Abcam, diluted 1:100) overnight at 4 °C. After the primary antibody incubation, the cells were incubated with Alexa Fluor-conjugated secondary antibodies (ab150105, Abcam) for 1 h at room temperature. Hoechst 33342 was used for counterstaining for 10 min. Finally, the slides were mounted using a mounting medium (S3023, Dako) and analyzed under a confocal microscope (LSM980, ZEISS).
Next, the cells were incubated with blocking buffer (3% BSA, 0.2% sodium azide, and 0.1% tween-20 in PBS) for 30 min at room temperature. Subsequently, the cells were incubated with primary antibody (ab110325, Abcam, diluted 1:100) overnight at 4 °C. After the primary antibody incubation, the cells were incubated with Alexa Fluor-conjugated secondary antibodies (ab150105, Abcam) for 1 h at room temperature. Hoechst 33342 was used for counterstaining for 10 min. Finally, the slides were mounted using a mounting medium (S3023, Dako) and analyzed under a confocal microscope (LSM980, ZEISS).
3
Blue Native Electrophoresis Analysis of Mitochondrial Supercomplexes
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Supercomplex levels and compositions were analyzed in isolated mitochondria from cells by BNE. Mitochondrial proteins were solubilized with 10% digitonin (4 g/g) (D5628, Sigma-Aldrich) and run on a 3 to 13% gradient blue native gel. The gradient gel was prepared in 1.5-mm glass plates using a gradient former connected to a peristaltic pump. Proteins were electroblotted onto polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-FL, 0.45 μm, Merck Millipore, IPFL00010) for 1 hour at 100 V in transfer buffer (48 mM tris, 39 mM glycine, 20% EtOH). A Mini Trans-Blot Cell system (Bio-Rad) was used. Sea Blocking buffer (37527, Thermo Fisher Scientific) or phosphate-buffered saline (PBS) with 5% BSA was used for 1 hour at room temperature (RT) to avoid nonspecific binding of antibodies. For protein detection, antibodies were incubated with the membrane for 2 hours at RT. Secondary antibodies were incubated for 45 min at RT. The membrane was washed with PBS–0.1% Tween 20 for 5 min three times between primary and secondary antibodies, and after secondary antibodies, the last wash was only PBS. To study supercomplex assembly, the PVDF membrane was sequentially probed with complex I (anti-NDUFA9, Abcam), complex IV (anti-COI, Invitrogen), and complex II (anti-SDHA, Thermo Fisher Scientific) antibodies.
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