After protein purification, protein concentration was determined using BCA protein reagents (Thermo Scientific). Samples were separated on a 4–12% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and probed with anti-Bcl-xL (1:1000 dilution, Cell Signaling), anti-ΔN-Bcl-xL (1:100 dilution, Aves Labs, Tigard, OR, USA), anti-active bax (1:100 dilution, Enzo Life Science, Farmingdale, NY, USA), anti-whole bax (1:1000 dilution, Cell Signaling), anti-cytochrome c (1:1000 dilution, Cell Signaling), anti-active caspase 3 (1:100 dilution, Abcam), anti-VDAC (1:1000 dilution, Cell Signaling), anti-COX IV (1:1000, Invitrogen) and anti-GAPDH (1:1000, Sigma-Aldrich). anti-ΔN-Bcl-xL is custom-produced (peptide sequence: CZ DSP AVN GAT GHS SSL D (1:100, Aves Labs). Membranes were treated with ECL reagents (Perkin Elmer) and images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Anti cox4
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Anti-COX4 is a laboratory reagent that detects the presence of the Cytochrome c Oxidase Subunit 4 (COX4) protein. COX4 is a component of the electron transport chain in mitochondria and is commonly used as a marker for mitochondrial content.
Automatically generated - may contain errors
Lab products found in correlation
Anti vdac, by Cell Signaling Technology (2 mentions)
Anti mtco1, by Thermo Fisher Scientific (2 mentions)
Bca protein reagent, by Thermo Fisher Scientific (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Anti active caspase 3, by Abcam (1 mentions)
Anti δn bcl xl, by Aves Labs (1 mentions)
Ecl reagent, by PerkinElmer (1 mentions)
Anti cytochrome c, by Cell Signaling Technology (1 mentions)
Anti bcl xl, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Iblot transfer system, by Thermo Fisher Scientific (1 mentions)
Western breeze, by Thermo Fisher Scientific (1 mentions)
Anti porin, by Thermo Fisher Scientific (1 mentions)
Ix71 system, by Olympus (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
N dodecyl β maltoside, by Merck Group (1 mentions)
Anti cox1, by Santa Cruz Biotechnology (1 mentions)
Anti cox 2, by Abcam (1 mentions)
Complete tablet, by Roche (1 mentions)
5 protocols using anti cox4
1
Western Blot Analysis of Apoptosis Markers
2
Mitochondrial Fractionation from Rat Brain
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Mitochondria or subfraction of mitochondria was purified from rat brain as previously described.10 (link), 19 (link), 56 (link) In brief, rats (n=12) were killed and brain tissue was homogenized in isolation buffer (250 mM sucrose, 20 mM HEPES, 1 mM EDTA, 0.5% BSA). After a series of centrifugations, the nuclear material and the mitochondrial pellet containing synaptosomes were separated. Synaptosomes were disrupted by applying 1200 psi pressure for 10 min and mitochondria were separated by ultracentrifugation. Subfractionation of mitochondria was performed as described previously.10 (link) In brief, the purified mitochondrial pellet was permeabilized and disassociated using 0.1% digitonin. Anti-VDAC (1:1000 dilution, Cell Signaling) was used as a marker for the outer membrane, and anti-COX IV (1:1000, Invitrogen) as a marker for the inner membrane to verify the quality of the fraction.
3
Quantitative Immunoblotting Analysis of Mitochondrial Complexes
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Electrophoresed gels were blotted onto polyvinylidene fluoride (PVDF) membranes using an iBlot transfer system (Invitrogen) according to the manufacturer’s instructions. Blotted PVDF membranes were blocked at room temperature for 30 min. Primary antibody probing was performed at room temperature for 90 min. Secondary antibody probing was performed with chromogenic antibody detection kit (WesternBreeze; Invitrogen) according to the manufacturer’s instructions. Primary antibodies used for SDS-PAGE were as follows: 0.5 μg/mL anti-porin (Molecular Probes), 2.5 μg/mL anti-MT-CO1 (Molecular Probes), 2.5 μg/mL anti-MT-CO2 (Molecular Probes), 2.5 μg/mL anti-COX4 (Molecular Probes), 2.5 μg/mL anti-COX5B (Molecular Probes). Primary antibodies used for BN-PAGE were as follows: 0.5 μg/mL anti-NDUFA9 for CI (Molecular Probes), 0.5 μg/mL anti-SDHA for CII (Molecular Probes), 0.5 μg/mL anti-UQCRC2 for CIII (Molecular Probes), 2.5 μg/mL anti-MT-CO1 for CIV (Molecular Probes), 0.5 μg/mL anti-ATP5B for CV (Molecular Probes).
4
Mitochondrial Protein Localization in Myoblasts
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Patient-derived myoblasts were seeded onto 4-well culture slides and were maintained at 37 °C under humidified atmosphere of 5 % CO2. After 3 days in culture, cells were fixed, permeabilized, and blocked according to standard immunocytochemical protocol. Primary antibody probing was performed at room temperature for 2 h. Secondary antibody probing was performed with 2.5 μg/mL Alexa Fluor 568 (Molecular Probes) at room temperature for 1 h. Mitochondria were co-stained with 0.25 μg/mL MitoTracker Green (Molecular Probes). Stained cells were observed under a fluorescent microscope (IX71 System; Olympus). Primary antibodies used were as follows: 2.5 μg/mL anti-MT-CO1 (Molecular Probes), 2.5 μg/mL anti-COX4 (Molecular Probes).
5
Whole Cell Lysate Preparation and Western Blotting
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Total cell lysates were prepared from cells as described previously [21] (link). Briefly, cells were washed twice with phosphate buffered saline pH 7.4 and suspended in lysis buffer (1.5% n-dodecyl β-maltoside (Sigma Aldrich, St. Louis, MO)) containing protease inhibitor tablets (Complete tablets; Roche, IN). Protein concentrations were measured by the DC Protein assay (Bio Rad, Hercules, CA). Equal amounts of protein were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gels followed by transfer to PVDF membranes. Membranes were stained with 0.1% Amido Black in 10% acetic acid for 2 minutes. Membranes were then probed with anti-Cox-1 1∶ 2000 (Santa Cruz Biotechnology Inc), anti-Cox-2 1∶ 1000 (Abcam, Cambridge, UK), anti-Cox-4 1∶ 8000 (Molecular Probes) and secondary antibodies from GE Health (IgG peroxidase linked species-specific whole antibody). Detection was performed by the enhanced chemical luminescence method (Pierce, Rockford, IL).
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