Palmitic acid (PA), stearic acid (SA), and fatty acid-free bovine serum albumin (BSA) were purchased from Sigma–Aldrich. C34, MG132 and actinomycin D were purchased from Caymen Chemicals. Antibodies against SREBP1 (ab28481), MCAD (ab92461), TLR4 (ab13556), TIRAP (ab17218), MyD88 (ab135693), and β-actin (ab6276) were purchased from Abcam. Antibodies against Sp1 (#9389), PU.1 (#2266), ACC (#3662), FASN (#3180), CPT1A (#97361), GLUT1 (#73015), p-NFκB (#3033), and β-catenin (#8480) were purchased from Cell Signaling. Horseradish peroxidase (HRP)-conjugated secondary antibodies (sc2357) were purchased from Santa Cruz Biotechnology. TLR4 promoter reporters were given as a kind gift by Professor Michael Rehli at the University of Regensburg Medical School, Germany. All the diets, including high-fat diet (HFD, D12492) and its matched control diet (CD, D12450J), Palmitic acid-rich diet (PAD, D16042106) and its matched control diet (C-PAD, D17042705), were purchased from Research Diets, Inc.
Ab92461
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab92461 is a lab equipment product offered by Abcam. It serves as a core function without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab188470, by Abcam (2 mentions)
Ab79822, by Abcam (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Palmitic acid, by Merck Group (1 mentions)
D12492, by Research Diets (1 mentions)
Ab28481, by Abcam (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Fatty acid free bovine serum albumin, by Merck Group (1 mentions)
Ab135693, by Abcam (1 mentions)
β actin ab6276, by Abcam (1 mentions)
Anti mouse na931, by GE Healthcare (1 mentions)
Ab14748, by Abcam (1 mentions)
Ab46798, by Abcam (1 mentions)
Na935, by GE Healthcare (1 mentions)
Sc 11414, by Abcam (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Ab196655, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab128568, by Abcam (1 mentions)
Ab57602, by Abcam (1 mentions)
14 protocols using ab92461
1
Investigating Lipid Metabolism Pathways
2
Western Blot Protein Analysis
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This work conducted WB assay in line with our previous study30 (link). For this, we electrophoresed proteins (50 µg) onto SDS-PAGE gel, followed by transfer onto PVDF membranes. Next, membranes were incubated with the ACADM (1:2000, Abcam, Britain, ab92461) or GAPDH (1:2000, Abcam, Britain, ab8245) antibodies and visualized them using ECL (Coolaber, Beijing, China), with GAPDH being the endogenous control.
3
Western Blot Analysis of Mitochondrial Proteins
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Each sample was loaded onto 4–20% Tris-glycine gels. After electrophoresis and transferring to nitrocellulose membranes, the membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 0.2% I-block (Tropix, T2015) for 90 min at room temperature. Membranes were then incubated overnight at 4 °C with following primary antibodies, anti-β-actin (1:1000, Sigma-aldrich A5441), anti-TOM40 (1:200, Santacruz, sc-11414), anti-ATP5A (1:500, Abcam, ab14748), anti-ACADM (1:500, Abcam, ab92461), and anti-HSP60 (1:500, Abcam, ab46798). After incubation with peroxidase-conjugated secondary antibodies, visualization was enhanced by chemiluminescence (GE Healthcare, NA931- anti-mouse, or NA934- anti-rabbit, or NA935- anti-rat). Optical density was assessed using the NIH Image analysis software.
4
Mitochondrial Protein Expression Analysis
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Fibroblasts grown in T75 flasks at 90–95% confluence were harvested by trypsinization, pelleted by centrifugation and stored at -80 °C. The BCA Protein Assay (Thermo scientific) quantified protein concentration. Proteins were separated on NUPAGE gels (Invitrogen) and transferred to nitrocellulose membranes. After 1 h of blocking, membranes were incubated with primary antibodies. The primary antibodies used as follows: VLCAD (GeneTex, Rb pAb GTX114232) 1:1000, MCAD (Abcam, Rb mAb ab92461) 1:1000, LCAD (Abcam, Rb mAb ab196655) 1:1000, TFPa (Abcam, Rb pAb ab54477) 1:500, TFPb (Bethyl, Rb A305-020A) 1:3000, CPT1α (Abcam, Ms mAb ab128568) 1:1000, MFN1 (Abcam, Ms mAb ab57602) 1:400, MFN2 (Abcam, Ms mAb ab56889) 1:400, DRP1 (Abcam, Rb mAb ab184247) 1:100 and GAPDH (Cell Signaling Technology, Rb mAb 2118S) 1:30000 dilutions overnight at 4 °C. The membranes were then incubated with fluorescent conjugated secondary antibodies for 1 h. The secondary antibodies used and their concentration as follows: Antibody DyLight 800 conjugated Anti-Rabbit IgG made in goat (611-145-002), Antibody DyLight 680 conjugated Anti-Rabbit IgG made in goat (611-144-003), Antibody DyLight 800 conjugated Anti-Mouse IgG made in goat (610-145-002), and Antibody DyLight 680 conjugated Anti-Mouse IgG made in donkey (610-744-124). Band signal intensities were obtained using Odyssey imaging system.
5
Western Blot Analysis of Liver Metabolic Proteins
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The liver tissues were lysed on ice using RIPA lysis buffer (Cell Signaling Technology, USA) supplemented with protease inhibitors and phosphatase inhibitors (Roche, USA), and 1 mM PMSF (Sigma, USA). Equal amount of proteins were separated on 10% SDS-PAGE gels. The tissues were then transferred onto nitrocellulose membranes (Bio-Rad, USA), and were subsequently blocked with 5% skimmed milk. The membranes were incubated with primary antibodies against PPAR-γ (ab45036, Abcam, U.S.), CPT-1 (ab104662 Abcam, U.S.), MCAD (ab92461, Abcam, U.S.) and GAPDH (G9545, Sigma-Aldrich, U.S.). After washing with 0.1% Tween 20 in TBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, U.S.) and bound antibody was detected by ECL Plus Reagents (Amersham Biosciences, U.S.). Bands were quantified and analyzed by Image Pro Plus software.
6
Quantitative Western Blot Analysis
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Total protein extracts were prepared from tissues and cells using RIPA buffer (Cell Signaling). Equivalent amounts of protein determined by BCA assay were resolved by 10% Bis-Tris gel electrophoresis and blotted to Immobilon PVDF membranes (Millipore). The following primary antibodies and dilutions were used: anti-DNMT1 (1:1000; ab13537, Abcam); anti-DNMT3a (1:1000; ab188470, Abcam); anti-DNMT3b (1:1000; ab79822, Abcam); anti-DNMT3L (1:1000; ab3493, Abcam); anti-BMP4 (1:1000; ab39973, Abcam); anti-TBX3 (1:1000; ab154828, Abcam); anti-ACADM (1:1000; ab92461, Abcam); anti-caspase 3 (1:1000; 9662S, Cell Signaling); anti-GAPDH (1:5000; 5174S, Cell Signaling). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Proteintech) was used as secondary antibody. The membranes were developed using Supersignal West Pico Chemiluminescent substrate (Thermo Scientific). To normalize sample loading and transfer, the ratio of band intensities to GAPDH was obtained to quantify the relative protein expression level.
7
Western Blot Analysis of Cardiac Proteins
8
Investigating Epigenetic Regulatory Proteins
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Antibodies against Piwil1, Piwil2, Stat1, Maoa, Acadm, Dnmt1, Dnmt3a and Dnmt3b were obtained from Abcam (catalogue numbers: ab12337, ab85084, ab92506, ab126751, ab92461, ab188453, ab188470 and ab79822, respectively). PCNA, cyclin A, cyclin D and cyclin E antibodies were obtained from Boster Biological Technology Co. Ltd. (catalogue numbers: BM1582, BM0104, BM4272 and BA0774, respectively). Decitabine, Azacitidine, Bortezomib (PS‐341) and MG‐132 were obtained from Selleck Chemicals (catalogue numbers: S1200, S1782, S1013 and S2619). All other reagents were from common commercial sources.
9
Western Blot Analysis of MCAD and GAPDH
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Western blotting was performed according to a previously described method [218 (link)]. GAPDH was used as a positive control. Commercially available antibodies were used to detect GAPDH (AB8245, Abcam, Cambridge, UK) and MCAD (AB92461, Abcam, Cambridge, UK). The original images can be found in Additional File 16 : Fig. S10.
10
Immunohistochemistry for Paraffin-Embedded Samples
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The paraffin-embedded specimens were cut into 4 µm thick section for immunohistochemical staining. The sections were manually dewaxed with xylene and washed in gradient alcohol and ddH2O. The sections were then placed in sodium citrate solution for antigen repair under high temperature and high pressure, and washed in ddH2O and PBS. The samples were incubated with 3% H2O2 at room temperature for 15 min and then washed again in PBS. Then 3% BSA was added and the samples were incubated at 37 °C for 30 min. Adding designated antibody (ab92461, Abcam, UK) and incubate overnight at 4 °C to avoid light. IHC was performed by SP Rabbit & Mouse HRP Kit(DAB) (CWBIO, China).
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