Western blotting was performed as previously described.21 (link) The following primary antibodies were used: APOC1 (1:1000, ab198288), cyclin B1 (1:5000, ab32053), cyclin D1 (1:5000, ab134175), caspase-9 (1:1000, ab202068), BCL-2 (1:500, ab32124), ERK1/2 (1:500, ab196883), phospho-ERK1/2 (1:500, ab65142), and GADPH (1:2500, ab9485) were purchased from Abcam Inc. Phospho-p38 (1:1000, #9211), p38 (1:1000, #9212), phospho-JNK (1:1000, #9251) and JNK (1:1000, #9252) were purchased from Cell Signaling Technology (Boston, MA, USA). Goat anti-rabbit IgG H&L (1:2000, ab6721, Abcam) was used as a second antibody.
Gadph
Manufactured by Abcam
Sourced in United States, United Kingdom, China
GAPDH is a glycolytic enzyme that catalyzes the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. It is a commonly used housekeeping gene for quantitative real-time PCR and Western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (4 mentions)
β actin, by Merck Group (3 mentions)
Mmp 9, by Abcam (3 mentions)
Cleaved caspase 3, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Occludin, by Abcam (2 mentions)
Hipure total rna mini kit, by Magen Biotechnology Co (2 mentions)
Cdna synthesis kit, by Takara Bio (2 mentions)
P stat3, by Abcam (2 mentions)
Traf6, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Tnf α, by Abcam (2 mentions)
Vimentin, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
Chemiluminescent detection system, by Bio-Rad (2 mentions)
Bca assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Gel doc ez imager, by Bio-Rad (2 mentions)
Ecl reagent, by Thermo Fisher Scientific (2 mentions)
46 protocols using gadph
1
Comprehensive Western Blotting Protocol
2
Comprehensive Protein Expression Analysis
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Approximately 100 mg of tissues was dissolved in T-PER Tissue Protein Extraction Reagent (including Protease Inhibitor Cocktail) for protein extraction. The concentrations of total protein were measured with BCA protein assay kit. Relative protein expressions were determined by WB according to the previous study. The primary antibodies used in this study were as follows: MUC2 (Novus NB120-11197, Littleton, CO, USA), ZO-1 (Thermo Fisher 40-2200, Waltham, MA, USA), Claudin1 (Abcam ab129119, Cambridge, UK), Occludin (Abcam ab222691, Cambridge, UK), GPR43 (Proteintech 19952-1-AP, Wuhan, China), GPR41 (Abcam ab103718, Cambridge, UK), HIF-1α (Thermo Fisher MA1-516, Waltham, MA, USA), AhR (Novus Biologicals NB100-2289, Littleton, CO, USA), IL-22 (Affinity DF8343, Changzhou, China) and GADPH (Abcam ab181602, Cambridge, UK). Relative levels of target protein were normalized against GAPDH.
3
Western Blot Quantification of Plasma Proteins
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For Western Blots, 1 µL of plasma or 20 µg of proteins was heated for 10 min at 80 °C with a buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). Samples were loaded and migration was achieved on 6% SDS-PAGE gels while protein transfer was performed on a nitrocellulose membrane (GE Healthcare Lifescience, Canada). The membrane was then blocked for 1 h in TBS-Tween buffer containing 5% of skimmed milk and washed in TBS-Tween buffer. Incubation was then achieved overnight with the appropriate primary antibody. The antibodies used are: apoB-48 primary antibody at 1/1000 in milk (apoB, MAB012, Millipore Sigma, USA), Plin-2 primary antibody at 1/250 in milk (Plin-2, 690102, Progen, Germany), ERK1/ERK2 primary antibody at 1/1000 in milk (ERK1/ERK2, 61–7400, ThermoFisher, Canada) and phospho-ERK1/ERK2 (phospho-ERK1/ERK2, MA5-15173, ThermoFisher, Canada). A specific secondary antibody conjugated with horseradish peroxidase was used to detect the relative amount of primary antibody. Bands were analyzed with the ImageLab software (Biorad). For apoB-48, total protein content was determined using stain-free gels (Bio-Rad, USA) and sample normalization was carried out using these values, as previously described69 (link). For Plin-2, β-actin (1:250 000, Sigma-Aldrich, USA) was used for sample normalization while GADPH (1:1000, Abcam, USA) was used for ERK and phosphoERK.
4
Comprehensive Lung and Atherosclerosis Study
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SIN‐1, ox‐LDL, HPF, and NAC were purchased from Sigma‐Aldrich. Lyso‐Tracker, ER‐Tracker, LDs‐Tracker, and Mito‐Tracker were purchased from Beyotime Biotechnology (Shanghai, China). Liposome synthesis material DSPE‐PEG2000‐NHS was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. Antibody iNOS and house keeping protein GADPH were brought from Abcam. Human nonsmall cell lung cancer cell line A549 and mouse monocyte macrophage leukemia cell line RAW 264.7 were obtained from the Shanghai Institutes for Biological Sciences (China). BALB/c nude mice (18 ± 2 g) and ApoE genetic defect mice (18 ± 2 g) are bought from Shanghai slaccas experimental animal Co., Ltd (Shanghai, China). All animals are Specific Pathogen Free and performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Xiamen University and the experiments were approved by the Animal Ethics Committee of the Xiamen University (32201145).
5
Quantifying Gene Expression in Osteosarcoma
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The normal human osteoblasts hFOB1.19, human osteosarcoma cells SJSA-1 and human osteosarcoma cells HOS used in this study were purchased from Shenzhen Aowei Biotechnology Co (http://www.otwobiotech.com/ ). We used normal human osteoblasts hFOB1.19 as normal control cells for osteosarcoma with reference to previous studies (22 (link), 23 (link)). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 100 U/mL penicillin, and 100 mg streptomycin at 37°C and 5% CO2 atmosphere. RNA extraction using Hipure Total RNA Mini kit (Magen, China) followed by quantitative real-time PCR (qRT-PCR) was used to purify the total intracellular RNA from the induced samples. Subsequently, 1000 ng of the extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Takara, China). qRT-PCR was used to detect the gene expression using the LightCycler 480 Sequence Detention System (Roche, Germany) and PCR Green Master Mix (Roche, Germany). The activation cycle of the polymerase included 10 min at 95°C and 15 s at 95°C, and 45 cycles such cycles were performed. Glyceraldehyde 3-phosphate dehydrogenase (GADPH, Abcam, USA) was used as the internal control, and the data analysis was performed using the 2–ΔΔCT method. The analysis for each gene was performed in triplicate. The primer sequences of the target genes are presented in Table 1
6
Western Blot Analysis of Complement Proteins
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Samples were lysed with RIPA buffer on ice and centrifuged at 16000 g for 15 min to extract protein. Protein concentrations were measured using the BCA Protein Assay kit (Thermo, 23,227). These protein samples were separated by electrophoresis using 8% or 15% SDS-PAGE at 100 V for 20 min and 120 V for 100 min. Proteins were electrostatically transferred to NC membrane and blocked with 5% BSA for 120 min. The primary antibodies were incubated overnight at 4°C and the secondary antibody for 60 min at room temperature. Finally, the labeled protein bands were developed with developing solution and scanned. All experiments were repeated three times. The antibodies used were as follows: GADPH (Abcam, 16,891; 1:1000), anti-CFH (Abclonal, A13686; 1:1000), anti-FHL (proteintech, 21,619-1-AP; 1:1000) and anti-α-SMA (Abcam, ab7817; 1:1000).
7
Chondroprotective Effects of Wogonoside
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Wogonoside (purity >98 %), was purchase from Nantong Feiyu Biological Technology Co, ltd (Nantong, China). Carboxymethylcellulose (CMC) and type II collagenases were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant human IL-1β was purchased from PeproTech (NJ, USA). The primary antibody against collagen II, aggrecan, MMP-9, MMP-13, ADAMTS5, collagen X, HIF-2α, TRAF6, p-ERK1/2, ERK1/2, p-Stat3, Stat3 and GADPH were acquired from Abcam (Cambridge, UK), MMP-3 and iNOS antibodies were obtained from Sigma-Aldrich (St Louis, MO, USA). Sox-9 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Anti-VEGF-A, Anti-Runx-2, Anti-IRAK1, goat anti-rabbit, and anti-mouse IgG-HRP was from Bioworld (OH, USA) and antibodies against COX-2, PI3K(p110), PI3K(p85), AKT, p-AKT, p-IKKα/β, IκBα, p-IκBα, p65, and p-p65 were purchased from Cell Signaling Technology (Danvers, MA, USA); Alexa Fluor®488 labeled and Alexa Fluor®594 labeled Goat Anti-Rabbit IgG (H+L) second antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). The 4', 6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime (Shanghai, China). The cell culture reagents were purchased from Gibco (Grand Island, NY, USA).
8
Polyclonal Antibodies for Proteomic Analysis
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The rabbit polyclonal antibody against APP was as described previously. Rabbit polyclonal anti-Nav1.6 (Alomone Laboratories), c-Jun N terminal protein kinase (JNK) (J4500; Sigma), p35 (cell signalling), F3 (Mellita), actin (abcam), TfR (Boster), GADPH (Abcam) and γ-tubulin (GTU88; Sigma) were obtained from the respective commercial sources. NF203 (Sigma), JNK inhibitor III (Calbiochem) and roscovitine (Calbiochem) were purchased.
9
Western Blot Analysis of Metabolic Regulators
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Tissues were homogenized and lysed by RIPA buffer. After centrifugation of the lysates at 12,000 g for 10 min at 4 degrees, the supernatants were collected. Equal amounts of protein were electrophoresed on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was then preincubated with blocking solution (PBST containing 5% fat-free milk) for 2h before incubation with primary antibodies at 4 degrees overnight. The following primary antibodies were used: GADPH (1:4000; Abcam), β-actin (1:4000; Abcam), AMPK, (1:2000; Abcam). p-AMPK (1:2000; Abcam), ACC (1:2000; Abcam), p-ACC (1:2000; Abcam), SIRT1(1:2000; Abcam), PGC1a (1:2000; Abcam), NF-kB (1:2000; Abcam). After washing with PBST, the blot was incubated with secondary antibody (HRP-conjugated, 1:10,000 dilution) for 90 min and then washed and detected by an enhanced chemiluminescence detection kit (Haigene).
10
Exosomal Protein Profiling by Western Blot
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Exosomal protein content was measured by BCA, and aliquots of 10 µg were used for Western blot analysis. Exosomes were lysed in Laemmli buffer at 95°C for 5 min, and the protein extracts were resolved by SDS-PAGE and probed using antibodies against HSP90 (1:1,000, SMC-107; Stressmarq Bioscience), TRP-2 (1:1,000; PEP8, kindly supplied by Dr. V.J. Hearing, National Institutes of Health, Bethesda, MD), β-actin (1:10,000, no. A5441; Sigma-Aldrich), GADPH (1:7,000, no. ab8245; Abcam), CD-9 (1:1,000, no. 92726; Abcam). Anti-CD63 and anti-CD81 hybridomas were kindly supplied by Dr. M. Yáñez-Mo (Molecular Biology Centre, Madrid, Spain). Peroxidase-conjugated AffinityPure donkey anti-rabbit or anti-mouse (1:5,000; Jackson ImmunoResearch) were used as secondary antibodies and their binding was detected using ECL Western Blotting Substrate kit (GE Healthcare). The intensity of the immunoreactive bands was quantified by densitometry using ImageJ software (National Institutes of Health).
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