All rats were sacrificed on day 21 of EAM. Total protein was prepared from the ventricle of rats. The cardiac ventricles were lysed in RIPA buffer (Product Code: P0013B, Beyotime, China) in the presence of 1 mmol/L phenylmethanesulfonyl fluoride (PMSF, Solarbio, China) and protein phosphatase inhibitor complex I (Aidlab, China), which contains sodium orthovanadate, sodium fluoride, sodium molybdate, sodium tartrate dihydrate, and imidazole. Protein samples were separated by 10% SDS-PAGE gels and were transferred to PVDF membranes (Millipore, United States). The membranes were blocked by 5% non-fat milk for 1 h and incubated with anti-connexin43 (1:10,000, Product Code: C6219, Sigma), anti-phospho-Cx43 (Ser368) (1:1,000, Product Code: 3511, Cell Signaling), anti-phospho-Cx43 (Ser262) (1:100, Product Code: sc-17219, Santa Cruz), anti-myristoylated alanine-rich C kinase substrate (MARCKS) (phospho S158) antibody (1:5,000, Product Code: ab81295, Abcam), and anti-GAPDH antibody (1:5,000, Epitomics). After rinsed with PBST for 5 times, the membranes were incubated with a goat anti-rabbit antibody (1:10,000, Sigma) for 1 h at room temperature. Immunoblots were developed using ChemiDoc™ Imaging Systems (Bio-Rad). Densitometry of bands was analyzed using ImageJ software (National Institutes of Health, United States).
Anti gapdh antibody
Manufactured by Abcam
Sourced in United States, United Kingdom, China
The Anti-GAPDH antibody is a primary antibody that specifically binds to the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein. GAPDH is a key enzyme involved in the glycolysis pathway and is often used as a loading control or reference gene in various biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (27 mentions)
Ripa lysis buffer, by Beyotime (17 mentions)
Polyvinylidene fluoride membrane, by Merck Group (12 mentions)
Anti bcl 2 antibody, by Abcam (7 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Polyvinylidene difluoride membrane, by Merck Group (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Ripa buffer, by Beyotime (6 mentions)
Anti akt antibody, by Cell Signaling Technology (6 mentions)
Anti e cadherin antibody, by Abcam (6 mentions)
Anti runx2 antibody, by Abcam (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Goat anti rabbit igg, by Abcam (5 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Anti bax antibody, by Abcam (4 mentions)
Anti vimentin antibody, by Abcam (4 mentions)
Image lab software, by Bio-Rad (4 mentions)
Anti β actin antibody, by Abcam (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
282 protocols using anti gapdh antibody
1
Cardiac Protein Expression Analysis
2
Protein Expression and Apoptosis Assay
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Antibodies against STAT3, p-STAT3 (Y705), P65, p-P65 (S536), AKT, p-AKT (S473), PARP, cleaved-caspase 3, cleaved-caspase 8 and cleaved-caspase 9 were obtained from Cell Signalling Technology. Anti-GAPDH antibody was from Epitomics. MK2206, GDC0941 and ABT263 were purchased from Selleckchem.
3
Cardiac Ventricle Protein Analysis
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The cardiac ventricles were lysed in RIPA buffer (Product Code: P0013B, Beyotime, China) in the presence of 1 mM phenylmethanesulfonyl fluoride (Solarbio, China) and protein phosphatase inhibitor complex I (Aidlab, China), which contains sodium orthovanadate, sodium fluoride, sodium molybdate, sodium tartrate dihydrate and imidazole. Protein samples were separated by 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). The membranes were blocked by 5% non-fat milk for 1 h and incubated with anti-caspase 3 antibody (Cell Signaling; 1:1000), anti-β-actin antibody (Immunoway; 1:2000), anti-PKC-α antibody (abcam; 1:4000), anti-GAPDH antibody (Epitomics; 1:5000). After washing with PBST for 5 times, the membranes were incubated with goat anti-rabbit antibody (Sigma; 1:10000) for 1 h at room temperature. Densitometry of bands was analyzed using Image J software (National Institutes of Health, USA).
4
Western Blot Analysis of Liver Proteins
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After the indicated animal experiments, liver tissues were lysed in ice-cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Nantong, China). Protein concentrations were determined with a BCA Protein Assay Kit (Pierce). Tissue lysates (50 μg protein) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred onto a polyvinylidene difluoride membrane. After blocking for 60 min at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk, the membranes were incubated overnight at 4°C with anti-pAkt 230:3 (Ser473) antibody (Cell Signaling, #9271), anti-Akt antibody (Cell Signaling, #9272), anti-pIRS-1 (Tyr632) antibody (Santa Cruz, sc-17196), or anti-GAPDH antibody (Epitomics, P04406). The membranes were washed thrice with Trisbuffered saline containing 0.1% Tween 2 and incubated with horseradish peroxidase-conjugated secondary antibodies, and then the protein levels were detected using an enhanced chemiluminescence kit (Millipore).
5
Immunoblot Analysis of Cell Signaling
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The total protein from cultured cells and tumors were obtained with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and separated by 10% polyacrylamide gel electrophoresis, followed by transferring onto polyvinylidene uoride membranes (Millipore, MA, USA). The protein membrane was incubated with primary antibodies (Anti-pERK1/2, anti-total ERK1/2, Anti-pMEK1/2, Anti-MEK1/2, and Anti-GAPDH antibody, Abcam, Cambridge, MA, USA) overnight at 4 °C. We used the enhanced chemiluminescence (Thermo Scienti c, Waltham, MA) to detect protein bands and semi-quanti ed the band density with an ImageJ analysis software (National Institutes of Health).
6
Calpain Inhibitor III Proteomic Analysis
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Calpain inhibitor III (MDL28170; CI III) were purchased from Calbiochem/Merck, dissolved in dimethylsulfoxide (DMSO) (Sigma-Aldrich) and stored at −20°C. The following antibodies against cellular structures were used: rabbit monoclonal anti-vimentin antibody (CST), anti-GAPDH antibody (Abcam); rat monoclonal anti-tubulin antibody (Abcam); mouse monoclonal anti-calpain 2 antibody (Abcam), anti-β-actin antibody (Abcam). Goat anti-rabbit, rabbit anti-rat and goat anti-mouse antibodies were from Abcam. Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore. The chemiluminescent Western blotting detection reagent ECL Plus was purchased from Thermo Scientific™. The Calpain Activity Assay Kit (Fluorometric) was from Abcam.
7
Angiogenic Protein Expression Analysis
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Immunoblotting for HIF-1α, VEGF, PDGF and PLGF was performed on day 7 after gene transfection. The immune-reactive band of different kDa was visualized in all protein preparations with an enzyme-linked chemiluminescence detection system (Amersham Pharmacia Biotech) and a mouse monoclonal anti-human HIF-1α antibody (1:1000; R&D), rabbit anti-VEGF antibody (1:2000, Abcam, Uk), rabbit anti-PDGF antibody (1:1000, Santacruz, USA) or rabbit anti-PLGF antibody (1:1000,Abcam, Uk) which shows cross-reaction with the above angiogenic genes respectively and normalized to anti-GAPDH antibody (1:10000, Abcam, UK).
8
Western Blot Analysis of Osteogenic Markers
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The cells were washed by cold PBS 3 times; then, 150 μL RIPA lysate (Beyotime Biotechnology, Shanghai, China) was added. The cells were lysed in ice water by ultrasound, and the protein content was determined by the Bradford method. An equal amount of proteins was taken from each group for 10% SDS-PAGE, and the proteins on the gel were transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked at 4°C for 1 h and then incubated at 4°C overnight with the following primary antibodies: anti-RUNX2 antibody (1:500; Abcam, USA), anti-Osterix antibody (1:1000; Abcam, USA), anti-OCN antibody (1:500; Abcam, USA), anti-PI3K antibody (1:1000; Abcam, USA), anti-Akt antibody (1:300; Abcam, USA), anti-p-PI3K antibody (1:500; Abcam, USA), anti-p-Akt antibody (1:100; Abcam, USA), and anti-GAPDH antibody (1:1000; Abcam, USA). After being cleaned twice with TBST, the membranes were incubated at room temperature for 1 h with fluorescein-labeled goat anti-rabbit IgG (ab205718, 1:2000). The membrane was visualized with an ECL detection kit (Millipore, Bedford, MA, USA) using a chemiluminescence imaging system (Millipore).
9
Protein Extraction and Western Blot Analysis
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Total protein from tissues and cell lines was extracted by using radioimmunoprecipitation assay lysis buffer (product no. P0013B; Beyotime Institute of Biotechnology). The total protein concentration was determined using the BCA method. A total of 30 µg of protein was separated by SDS-PAGE (10%) and transferred to a PVDF membrane. Non-specific binding sites were blocked by incubating with TBST (0.1% Tween 20) containing 5% (w/v) non-fat dried milk for 1 h at room temperature. The antibodies used were as follows: Anti-SOX12 primary antibody (product no. SAB1412152; dilution, 1:1,000; Sigma-Aldrich; Merck KGaA), anti-JAK primary antibody (product code ab108596; dilution, 1:5,000), anti-phosphorylated (p)-JAK at Tyr1007+1008 primary antibody (product code ab32101; dilution, 1:2,000), anti-STAT3 primary antibody (product code ab68153; dilution, 1:1,000), anti-p-STAT3 (phosphorylated at Tyr705) antibody (product code ab76315; dilution, 1:5,000), anti-GAPDH antibody (product code ab9482; dilution, 1:5,000; all from Abcam) and goat anti-rabbit immunoglobulin G H&L (cat. no. ab205718; dilution, 1:5,000; Abcam). Signals were visualized by ECL chemiluminescence (cat. no. 34095, Thermo Fisher Scientific, Inc.). The results were evaluated with Quantity One software (v4.6.6; Bio-Rad Laboratories, Inc.).
10
Western Blot Analysis of IGFBP5, ERK, and Apoptosis Markers
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According to standard methods, Western blot analysis was performed. Briefly, proteins were separated on 10% SDS‐PAGE gels and then transferred to PVDF membranes (Amersham). The membranes were blocked using 5% nonfat dried milk for 2 hours and then incubated for 12 hours with an anti‐IGFBP5 antibody (1:1000; Abcam), anti‐ERK antibody (1:1000; Abcam), anti‐pERK (phosphorylated ERK) antibody (1:500; Abcam), anti‐Bax antibody (1:1000; Abcam), anti‐caspase‐3 antibody (1:1000; Abcam), anti‐Bcl2 antibody (1:1000; Abcam) or anti‐GAPDH antibody (1:10 000; Abcam). After washing in TBST (10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl and 0.1% Tween 20), the membranes were incubated for 2 hours with a goat anti‐rabbit antibody (1:5000; Abcam). Normalization was performed by blotting the same membranes with an antibody against GAPDH.
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