Western blotting analysis was performed as detailed previously.32 (link) Antibodies used in this study include anti-YARS2 (ab228957, at a dilution of 1:1000), anti-MT-ND1 (ab181848, at a dilution of 1:2000), anti-MT-ND5 (ab230509, at a dilution of 1:1000), anti-MT-CO2 (ab79393, at a dilution of 1:5000), and anti-TOM20 (ab186735, at a dilution of 1:2000) from Abcam, anti-MT-ND6 (PA5-43532, at a dilution of 1:1000) and anti-MT-ND4 (PA5-97298, at a dilution of 1:1000) from ThermoFisher, anti- MT-CYTB (55090-1-AP, at a dilution of 1:1000) from Proteintech, anti-β-actin (AF5003, at a dilution of 1:2000) from Beyotime Institute of Biotechnology, and HRP-conjugated anti-rabbit secondary antibody (A0208, at a dilution of 1:1000) from Beyotime Institute of Biotechnology. Immunoreactive proteins were visualized using a chemiluminescent immunodetection system (ChemiDoc XRS). Image J was employed to analyze the grayscale values obtained by western blotting.
Ab79393
Manufactured by Abcam
Sourced in China, United Kingdom
Ab79393 is a primary antibody that recognizes the protein of interest. It is a high-quality research-use tool designed for investigating the target protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Mirax scan, by Zeiss (2 mentions)
Dab eqv peroxidase, by Vector Laboratories (2 mentions)
Ab14705, by Abcam (2 mentions)
Ab92624, by Abcam (2 mentions)
Caseviewer, by 3DHISTECH (2 mentions)
Anti β actin af5003, by Beyotime (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Ab186735, by Abcam (1 mentions)
Ab181848, by Abcam (1 mentions)
Ab230509, by Abcam (1 mentions)
Anti gapdh antibody, by Proteintech (1 mentions)
Ab110272, by Abcam (1 mentions)
Sc 130301, by Santa Cruz Biotechnology (1 mentions)
Sc 33129, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Mitochondrial lysate, by Beyotime (1 mentions)
Ab0037, by Abcam (1 mentions)
Ab178945, by Proteintech (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
7 protocols using ab79393
1
Mitochondrial Protein Analysis by Western Blot
2
Protein Expression Analysis Protocol
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Protein extraction was carried out using the RIPA buffer, and the BCA protein assay Kit was used to quantitate total protein levels. Protein (40 μg per lane) was separated by SDS-PAGE. Proteins were transblotted to PVDF membranes, and membranes were blocked in 5% nonfat milk powder and incubated overnight at 4 °C with anti-FLAG (1:1000; CST, 14793S), anti-TFB1M (1:1000; Sigma-Aldrich, HPA029428), anti-TFB1M (1:1000; Abcam, ab236901), anti-MT-CO2 (1:1000; Abcam, ab79393), Bcl-2 (1:1000, Abcam, ab32124), Cleaved Caspase-3 (1:1000, Affinity, AF7022), E-cadherin (1:1000, CST, 3195 T) and N-cadherin (1:1000, CST, 13116 T). After incubated with horseradishperoxidase-conjugated goat anti-rabbit IgG, membranes were resolved by chemiluminescence. All membranes were stripped and reprobed with anti-GAPDH antibody (1:8000, Proteintech, China) as a loading control.
3
Protein Expression Quantification in HUVEC
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Total protein for AZU detection was extracted from the cytoplasm of the HUVECs, while for Cox II expression determination, specimens were extracted from the mitochondrial matrix. Total proteins from the cells were extracted using cytoplasmic protein reagent A (Beyotime Institute of Biotechnology), while total proteins from the mitochondrial matrix were extracted using a mitochondrial lysate (Beyotime Institute of Biotechnology). The proteins were quantified using the Bradford method. Samples of 50 μg protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with antibodies against Cox II (ab79393; 1:100) and Cox IV (ab110272; 1:100; Abcam, Shanghai, China), AZU (sc-33129; 1:100) and β-actin (sc-130301; 1:500; Santa Cruz Biotechnology, Inc. Dallas, TX, USA). Following incubation with a peroxidase-coupled anti-mouse IgG (sc-2371; Santa Cruz Biotechnology, Inc.) at 37°C for 2 h, bound proteins were visualized using enhanced chemiluminescence (Pierce Biotechnology, Inc., Rockford, IL, USA) and detected using a BioImaging System (UVP, Inc., Upland, CA, USA). Relative protein expression levels were quantified using β-actin and Cox IV as the loading controls.
4
Histological Analysis of Dissected Brains
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Dissected brains were fixed in 10% neutral buffered formalin overnight and mounted into paraffin blocks; 5‐μm sections were stained using routine histology techniques. Antibodies used for immunostaining were chicken anti‐GFP (Aves, #1020), anti‐MTND5 (Abcam, #ab92624), anti‐MTCO1 (Abcam, #ab14705), and anti‐MTCO2 (Abcam, #ab79393). Secondary antibodies were conjugated with horseradish peroxidase, and DAB EqV Peroxidase was used as a substrate (all Vector Laboratories). Slides were scanned with MiraxScan (Zeiss), and images were analyzed using CaseViewer (v2.2, 3DHistech) and Fiji/ImageJ (v1.51j, NIH).
5
Western Blot Analysis of Inflammatory Markers
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Cells or colon tissue samples were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors for 30 min on ice. The homogenates were centrifuged at 11,000 rpm for 20 min at 4 °C. The total protein concentrations in the supernatants were quantified by the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples with equal concentrations were separated in SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad). After blocking with 5% BSA for 2 h, the membranes were incubated at 4 °C overnight with primary antibodies against the following target proteins: TNF-α (ABclonal, A11534, dilution 1:1000), IL-6 (ABclonal, A0286, dilution 1:1000), IL-1β (ABclonal, A16288, dilution 1:1000), COX2 (Abcam, ab79393, dilution 1:1000), iNOS (Abcam, ab178945, dilution 1:1000), β-Actin (Proteintech, CL594-66009, dilution 1:5000), GAPDH (Abways, AB0037, dilution 1:5000), Claudin-2 (Abcam, ab53032, dilution 1:1000), Claudin-4 (Abcam, ab15104, dilution 1:1000), Claudin-7 (Abcam, ab27487, dilution 1:1000), ZO-1 (Abcam, ab96587, dilution 1:1000), and Occludin (Abcam, ab216327, dilution 1:1000). After washing, the membranes were incubated with the corresponding secondary antibodies for 2 h at room temperature (RT). The immune complexes were detected with an ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA).
6
Histological Analysis of Dissected Brains
7
Mitochondrial Protein Profiling by Western Blot
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Snap-frozen cell pellets (~50 mg) were homogenized in 5-fold volume extraction buffer (20 mM Tris-HCl, pH 7.6, 250 mM sucrose, 40 mM KCl, 2 mM EGTA). The post-nuclear supernatant (600 g homogenate) containing the mitochondrial fraction was used for western blot analysis with the following antibodies: Anti-VDAC1 (1:2000, ab15895; Abcam, Cambridge, UK), Anti-NDUFS4 (1:1500, ab55540; Abcam), Anti-SDHA (1:2000, ab14715; Abcam), Anti-UQCRC2 (1:1500, ab14745; Abcam), Anti-MTCO2 (1:2000, ab79393; Abcam), Anti- ATP5A (1:2000, ab110273; Abcam), and Anti-Vinculin (1:10,000, ab129002; Abcam). The visualized bands were quantified with Bio-Rad’s Image Lab software. The intensity of the bands was normalized to that of vinculin.
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