Tissue proteins were extracted using RIPA buffer with freshly added proteinase inhibitors. Protein concentrations were determined using BCA Protein Assay Kit (Pierce). Equal amounts of protein samples were subjected to western blot. The following antibodies were used: anti-UCP1 (Abcam, ab209483, 1:5000 dilution), anti-PGC-1a (Bioworld, BS72263, 1:500 dilution), anti-PER-ILIPIN (Cell Signaling Technology, 9349T, 1:1000 dilution), anti-PPARγ (CusAb, CSB-PA018424LA01HU, 1:500 dilution), anti-COX4 (Proteintech, 11242-1-AP, 1:1000 dilution), and anti-ACTIN (Sigma, A5441, 1:5000 dilution). Densitometry was performed using Image J. Relative band density was calculated by dividing the densitometry of target protein with loading control from the same membrane.
Anti cox 4
Manufactured by Proteintech
Sourced in China, United States
Anti-COX IV is a primary antibody product used for the detection of Cytochrome c Oxidase Subunit IV (COX IV) in various research applications. COX IV is a subunit of the cytochrome c oxidase complex, which is a key enzyme in the mitochondrial electron transport chain. This antibody can be used for techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to identify and localize COX IV in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti calreticulin, by Cell Signaling Technology (2 mentions)
Anti pkm1, by Cell Signaling Technology (2 mentions)
Anti p70 s6 kinase, by Cell Signaling Technology (2 mentions)
Anti pkm, by Abcam (2 mentions)
Anti pkm2, by Cell Signaling Technology (2 mentions)
Anti human specific ldha, by Cell Signaling Technology (2 mentions)
Anti ldhb, by Abcam (2 mentions)
Anti human and rabbit ldha, by Abcam (2 mentions)
Anti golgin, by Cell Signaling Technology (2 mentions)
Anti gpt2, by Santa Cruz Biotechnology (2 mentions)
Anti phgdh, by Merck Group (2 mentions)
Anti pc, by Santa Cruz Biotechnology (2 mentions)
Pi31430, by Thermo Fisher Scientific (2 mentions)
Pi 31460, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti actin, by Merck Group (1 mentions)
Anti ucp1, by Abcam (1 mentions)
Anti perilipin, by Cell Signaling Technology (1 mentions)
Anti opa1, by BD (1 mentions)
Anti tom20, by Proteintech (1 mentions)
14 protocols using anti cox 4
1
Western Blot Analysis of Adipocyte Proteins
2
Western Blot Analysis of Mitochondrial Proteins
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Cells were harvested by 0.25% trypsin (Gbico), cell suspension was then centrifuged, pellet cells were resuspended with 1× sample buffer, and boiled for 12 min. The protein samples were separated by SDS-PAGE and transferred into 0.45 μm PVDF membranes, 2 h later, membranes were blocked by 5% non-fat milk for 1 h, then incubated with primary antibody 30 min-2h in 5% non-fat milk followed by incubation with 1:5000 HRP secondary antibodies for 1 h. Detection of immunoreactive protein on Fuji x-ray film using ECL chemiluminescent substrate (Bio-Red). The following primary antibodies were used: anti-GAPDH and anti-Beta-Tubulin were obtained from Santa Cruz Biotechnology; anti-COX4, anti-COX2, anti-ATAD3A, anti-Mic60, anti-Yme1L, and anti-Tom20 were from Proteintech; anti-OPA1 was obtained from BD Biosciences, anti-Sam50 was obtained from Abcam; anti-Mic19 was from Abclonal; anti-Mic10 was from Invitrogen.
3
Protein Detection Reagents for Western Blotting and Immunofluorescence
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Detecting reagents used for western blotting include: streptavidin-HRP (Cell Signaling, 3999S, 1:3000), anti-Myc (Cell Signaling, 2276S, 1:3000), anti-V5 (Abcam, ab27671, 1:3000), anti-β actin (GenScript, A00702, 1:3,000), anti-FLAG (GNI, GNI14110-FG, 1:3000), anti-ABCD3 (Sigma, P0497, 1:3000), anti-MFN1 (Cell Signaling, D6E2S, 1:3000), anti-MFN2 (Cell Signaling, D1E9, 1:3000). Antibodies used for immunofluorescence include: anti-MFN1 (Cell Signaling, D6E2S, 1:500), anti-MFN2 (Cell Signaling, D1E9, 1:500), anti-calnexin (Abcam, ab22595, 1:500), anti-EEA1 (Cell Signaling, C45B10, 1:500), anti-GM130 (Abcam, ab52649, 1:500), anti-LAMP1 (Cell Signaling, D2D11, 1:500), anti-ABCD3 (Sigma, SAB4200181, 1:500), anti-ABCD3 (Sigma, P0497, 1:500), anti-catalase (Merck/Millipore, 219010, 1:500), anti-FLAG (GNI, GNI14110-FG, 1:500), anti-FLAG (Proteintech, 20543-1-AP, 1:500), anti-Myc (Cell Signaling, 2276S, 1:500), anti-COX4 (Proteintech, 11242-1-AP, 1:500).
4
Mitochondrial Protein Isolation and Analysis
5
Western Blot Analysis of Metabolic Enzymes
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Cell lysates were prepared and subjected to WB analysis as performed previously51 (link),52 (link). The following commercial antibodies were used: anti-β-actin (Sigma A1978), anti-PKM2 (Cell Signaling Technology 3198S), anti-PKM1 (Cell Signaling Technology 7067), anti-PKM (Abcam AB118499), anti-human specific LDHA (Cell Signaling Technology 2012S), anti-LDHB (Abcam AB75167), anti-human and rabbit LDHA (Abcam AB135396), anti-p70 S6 kinase (Cell Signaling Technology 2708T), anti-calreticulin (Cell Signaling Technology 12238T), anti-golgin (Cell Signaling Technology 13192), anti-COX IV (Proteintech 11242-1-AP), anti-GPT2 (Santa Cruz sc-398383), anti-PHGDH (Sigma HPA021241), and anti-PC (Santa Cruz sc-271493), horseradish peroxidase-conjugated secondary antibodies anti-mouse (Fisher PI31430) and anti-rabbit (Fisher PI31460). All the primary antibodies were used at a 1:500 dilution in 5% non-fat milk in TBST. Secondary antibodies were used at a 1:3000 dilution in 5% non-fat milk in TBST.
6
Western Blot Analysis of Metabolic Enzymes
7
Protein Expression Analysis of Mouse Brain Tissues
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Brain tissues were harvested from the sensorimotor cortex of the mice 3 days after SAH. The tissues were lysed with RIPA assay buffer, and 20 µg of proteins were separated via 4–20% SDS–PAGE and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated overnight with the indicated primary antibodies at 4 °C. The following primary antibodies were used: anti-β-actin (1:10,000; Proteintech), anti-TauT (1:1000; Abcam), anti-bax (1:1000; Proteintech), anti-bcl-2 (1:1000; Proteintech), and anti-COX IV (1:1000; Proteintech) antibodies. Chemical reactions were detected with an ECL system. The scanned images were analyzed with ImageJ software.
8
Immunostaining of FLAG and COXIV in Cells
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Cells were fixed with 3.7% formaldehyde, permeabilized with 1% Triton X-100, and blocked with 20% EzBlock Chemi (ATTO). Immunostaining was performed with the following primary antibodies: anti-FLAG (1:1000, mouse clone 1E6, 014-22383, Fujifilm) and anti-COXIV (1:100, rabbit polyclonal, 11242-1-AP, Proteintech). Antimouse IgG Alexa fluor 594 (1:1000, A11005, Invitrogen) or antirabbit IgG Alexa fluor 488 (1:1000, pA11008, Invitrogen) was used as a secondary antibody. To visualize DNA, the cells were stained with DAPI (1:1000). Images were acquired using a DMI 6000 B (Leica).
9
Immunostaining for Autophagy and Lysosomal Markers
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Immunostaining was performed as described elsewhere, particularly in [19 (link)]. The following primary antibodies:anti-Lamp2 (1:100, Proteintech, Beijing, China), anti-LC3 (1:100, CST, Beverly, MA, USA), anti-p62 (1:100, CST, Beverly, MA, USA), anti-Cathepsin B (CTSB) (1:100, CST, Beverly, MA, USA) and anti-Cathepsin D (CTSD) (1:100, Proteintech, Beijing , China) and anti-Parkin (1:100, Proteintech, Beijing, China) and anti-COX IV (1:100, Proteintech, Beijing, China).
10
Immunoblotting Analysis of Mitochondrial Proteins
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Extracts for immunoblotting were acquired from N2a cells homogenized in RIPA buffer (radioimmunoprecipitation assay buffer, Thermo Fisher Scientific, Catalog Numbers 89900). Briefly, proteins were separated by electrophoresis and subsequently transferred to the PVDF membrane by electroblotting using standard protocols. The primary antibodies used were anti-LC3II (Cell Signaling Technology, catalog #2775s), anti-COX IV (Proteintech, catalog no. 11242–1-AP), anti-TOM40 (Proteintech, catalog no. 18409–1-AP), anti-MnSOD (Proteintech, catalog no. 24127–1-AP), anti-CLS (Proteintech, catalog no.14845–1-AP), anti-PLS3 (Proteintech, catalog no.28028–1-AP), anti-NDPK-D (Bioss, bs-11902R), anti-PINK1 (Novus Biologicals, catalog no. BC100–494), anti-Parkin (Abcam, catalog no. ab77924), anti-Drp1 (Cell Signaling Technology, catalog #8570), anti-Lamp2 (Cell Signaling Technology, catalog #49067), PGC1a monoclonal antibody (Proteintech, catalog no. 66369–1-Ig), recombinant anti-mtTFA antibody (Abcam, ab252432), GAPDH monoclonal antibody (Proteintech, catalog no. 60004–1-Ig), and anti-Tubulin (Proteintech, catalog no. 11224–1-AP).
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