The primary antibodies used in this study were as follows: MitoProfile Total OXPHOS Rodent WB Antibody Cocktail [NADH dehydrogenase (ubiquinone) 1β subcomplex 8 (NDUFB8), succinate dehydrogenase complex subunit B (SDHB), ubiquinol-cytochrome c reductase core protein II (UQCRC2), ATP synthase, H+ transporting, mitochondrial F1 complex, α-subunit (ATP5A), ab110413, Abcam], NADH dehydrogenase (ubiquinone) iron-sulfur protein 4 (NDUFS4; ab139178, Abcam), cytochrome c oxidase subunit IV (COX IV; ab14744, Abcam), and CS (ab129095; Abcam). The following secondary antibodies were used in the present study: rabbit anti-goat IgG (H&L) (#A102PT; American Qualex, San Clemente, CA, United States ) and mouse anti-goat IgG (H&L) (#A106PU; American Qualex).
Ab14744
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab14744 is a primary antibody that targets a specific protein. It can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Ab14745, by Abcam (7 mentions)
Ab14715, by Abcam (5 mentions)
Ab14748, by Abcam (4 mentions)
Ab14705, by Abcam (4 mentions)
Ab14713, by Abcam (3 mentions)
Ab14711, by Abcam (2 mentions)
A1978, by Merck Group (2 mentions)
Ab7291, by Abcam (2 mentions)
Ab110242, by Abcam (2 mentions)
Ab14714, by Abcam (2 mentions)
Ab110258, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Ab9484, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Ab54481, by Abcam (2 mentions)
Ab15895, by Abcam (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Mitoprofile total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
33 protocols using ab14744
1
Mitochondrial Protein Quantification Protocol
2
Mitochondrial Protein Complex Analysis
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Mitochondrial pellets were solubilized with digitonin at 4:1 g protein and fractionated in blue native (BN) gels [28 (link)]. A total of 30 μg of mitochondrial proteins were fractionated in NativePAGE 3–12% Bis-Tris Protein Gels, 1.0 mm, 10-well (Invitrogen) at 4°C using cathode (50 mM Tricine, 15 mM Bis-Tris, pH 7.0 and 0.02% Serva blue G) and anode buffers (50 mM Bis-Tris, pH 7.0). Electrophoresis was carried out at a constant voltage (70 V) until the samples entered the polyacrylamide gradient (approximately 30 minutes) and then at a constant current (15 mA) until the dye reached the end of the gel (approximately 1 hour). After fractionation, the gels were electroblotted onto PVDF membranes and processed for immunoblot analysis as described above. The following primary antibodies were used: mouse anti-NADHs9 (NDUFA9, clone 15/22-5 [89 (link)], 1:1,000), mouse anti-NDUFS3 (clone 17D95, Abcam, ab14711, 1:1,000), mouse anti-NDUFS4 (clone 2C7CD4AG3, Abcam, ab87399, 1:1,000), mouse anti-SDH-A (clone 2E3GC12FB2AE2, Abcam, ab14715, 1:1,000), mouse anti-UQCRC2 (clone 13G12AF12BB11, Abcam, ab14745, 1:1,000), mouse anti-MT-CO1 (clone 1D6E1A8, Invitrogen, #459600, 1:1,000), mouse anti-MT-CO3 (clone DA5BC4, Abcam, ab110259, 1:1,000), mouse anti-COX IV (clone 20E8C12, Abcam, ab14744, 1:1,000), and rabbit anti-β-F1 [93 (link)] (1:20,000).
3
Western Blot Analysis of Skeletal Muscle
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Skeletal muscle and fibroblast homogenates were obtained according to previously described methodologies.30 (link) 30–40 μg (S1–S3) and 20 μg (S4) of whole-cell protein extracts were separated by SDS polyacrylamide (12%) electrophoresis and then wet transferred to polyvinyl difluoride (PVDF) membranes. For S4, a 4%–12% gradient gel was used. Immunological detection of proteins was carried out with the following primary antibodies: C1QBP (ab24733, Abcam), β-actin (A1978, Sigma), α-tubulin (ab7291, Abcam), and OXPHOS complex-specific antibodies (NDUFS3 [ab14711, Abcam], NDUFB8 [ab110242, Abcam], NDUFA9 [MS111, Molecular Probes], SDHA [459200, MitoSciences], SDHB [ab14714, Abcam], UQCRC2 [ab14745, Abcam], COXI [ab14705, Abcam], COXII [ab110258, Abcam], COXIV [ab14744, Abcam], and ATP5A [ab14748, Abcam]). Species-appropriate horseradish-peroxidase-conjugated secondary antibodies (DAKO, P0399, and P0260) were used.
4
Immunofluorescence Imaging of Ovarian Tissue
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Wild type ovaries (6 weeks) were fixed in 2.5% glutaraldehyde, 2% paraformaldehyde, 0.15 M sodium cacodylate buffer, and 0.1 M sucrose. Ovaries were embedded in paraffin, sectioned at 7 µm, and mounted on slides. Slides were rehydrated in 100% xylene, graded ethanols, washed in PBS, 0.01 M sodium citrate at 95 °C for 20 min, and room temperature sodium citrate for 20 min. Slides were washed 2x in ddH2O for 2 min and 3x in PBS for 3 min, and blocked in incubation buffer (1% BSA, 10% fetal calf serum, 0.1% Tween-20 in PBS) for 1 hour at room temperature. Slides were then incubated in 0.1% Sudan Black B (Sigma Aldrich, St. Louis, MO) in 70% ethanol for 20 min, washed 3x in PBS for 5 min, and jet washed with PBS. Primary antibodies mouse monoclonal anti-COX IV (abcam ab14744, 1:200) and goat polyclonal anti-P-gp (Santa Cruz sc-1517, 1:200) overnight at 4 °C in a humidity chamber, and secondary antibodies fluorescent red anti-mouse (1:200) and green anti-goat (1:200) for 1 hour at room temperature were used. Samples were washed and mounted with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA) and imaged with a confocal laser scanning microscope (upright Zeiss LSM800, Carl Zeiss Inc, Thornwood, NY).
5
Visualizing Mitochondrial-Autophagy Interplay
6
Mitochondrial Protein Analysis by Immunoblotting
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Immunoblotting was performed using 4–12% gradient gels through MOPS SDS-PAGE, as previously described [54 (link), 55 (link), 57 (link)–60 (link)]. Normalization of protein content was assessed using the Bradford Method [56 (link)]. Primary antibodies utilized in the study included the following: total OXPHOS Blue Native WV Antibody Cocktail (ab110412) (anti-NDUFA9 (complex I, ab14713, Abcam, Cambridge, MA), anti-SDHA (complex II, ab14715, Abcam), anti-UQCRC2 (complex III, ab14745, Abcam), anti-COX IV (complex IV, ab14744, Abcam), and anti-ATP5A (complex V/ATP Synthase, ab14748, Abcam)), anti-ATP5F1 (complex V/ATP Synthase, ab117991, Abcam), and anti-VDAC (#4866, Cell Signaling Technology, Danvers, MA). Goat anti-mouse IgG (H&L) horseradish peroxidase (HRP) conjugate 1:10,000 (Thermofisher Scientific) and goat anti-rabbit IgG HRP conjugate 1:5000 (Abcam) were used as the secondary antibodies. Normalization of protein content was through VDAC expression. Chemiluminescence quantified with Radiance Chemiluminescent Substrate (Azure Biosystems, Dublin, CA), per manufacturer’s instructions and imaged using the G:Box Bioimaging system (Syngene, Frederick, MD). GeneSnap/GeneTools software (Syngene) was used to acquire images. Densitometry was analyzed using Fiji Software (NIH, Bethesda, MD).
7
Evaluating mitochondrial complexes by BNGE
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Blue-native gel electrophoresis (BNGE) on fibroblast (P1 and P3) and muscle (P2 and P3) samples was performed as previously described.12 (link),13 (link) Mitochondria were solubilized with either n-dodecyl-β-d-maltoside (DDM), 1.6 mg/mg of mitochondrial protein or digitonin 4 mg/mg of mitochondrial protein.13 (link) Samples were run on precast native polyacrylamide 3%–12% Bis-Tris gels. Proteins were either transferred on a nitrocellulose membrane (1D-BNGE) or denatured and run on SDS-PAGE (2D-BNGE). Anti-ATP5A (Abcam, ab14748), anti-ATP6 (Abcam ab219825), and anti-COXIV (Abcam, ab14744) antibodies were used for complex V and complex IV visualization.
8
Immunoprecipitation and Western Blotting Assay
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Assays were performed as described previously (Han et al., 2015 (link); Chen et al., 2017 (link)). In brief, cells were lysed in cold cell lysis buffer (HEPES pH 7.4 50 mmol/L, NaCl 150 mmol/L, Triton X-100 1% and glycerol 10%) supplemented with phosphatase and protease inhibitors (B14011 and B15001, Bimake), sonicated and centrifuged for 15 min at 15,000 rpm at 4 °C. Total protein (20 μg) from each lysate was separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with the indicated antibodies. For immunoprecipitation, control IgG or antibody was incubated overnight at 4 °C with supernatant. Antibodies were purchased as follows: mouse monoclonal anti-HA antibody (MMS-101P), Covance; mouse monoclonal anti-FLAG antibody (F1804) and anti-Tubulin (T5201), Sigma-Aldrich; rabbit polyclonal anti-HA (561) and anti-FLAG (PM020), MBL International; anti-MFN2 (ab56889) and anti-COX4 (ab14744), Abcam; anti-PKM2 (4053), anti-S6K (2708s) and anti-pS6K (9234), Cell Signaling Technology; anti-Myc (SC-40), Santa Cruz. The phospho-S200 MFN2 antibody was generated and purified by AbMax Biotechnology.
9
Protein expression analysis in tissues
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Whole ovaries, livers, and kidneys and mitochondrial isolates were run on a western blot and incubated with mouse monoclonal anti-COX IV (abcam ab14744, 1:1000), mouse monoclonal anti-Actin (abcam ab8226, 1:1000), and rabbit monoclonal anti-P-gp (abcam ab170904, 1:1000) primary antibodies in 2% BSA overnight at 4 °C. Blots were labeled with corresponding rabbit anti-mouse or goat anti-rabbit HRP-conjugated secondaries, (1:3000) for 1 hour. HRP-labeled proteins were developed in ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA).
10
Comprehensive Protein Expression Analysis
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Western blot analysis was performed to detect the related protein levels [19 (link),20 (link)]. Tissues or cells were homogenized, boiled, separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. Blots were probed with appropriate primary antibodies against FAM3A (MA5-24107, ThermoFisher; HPA056991, SAB4300968, Sigma), HIF1α (ab51608, Abcam), VEGFA (ab51745, Abcam), CREB (9197, Cell Signalling Technology (CST)), pCREB (Ser133) (9198, CST), COXIV (ab14744, Abcam), PPARγ (ab45036, Abcam), Flag (ab205606, Abcam) and β-actin (3700, Cell Signalling Technology) at 4 °C overnight. After washing, the membrane was incubated with the appropriate secondary antibodies. Image signals were detected using Image Lab software.
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