40 μg proteins were separated by 10 % sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE), and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the primary antibodies against Histone H1 (Millipore, 1:1000), Non-phospho (Active) β-catenin (CST, 1:1000), β-catenin (CST, 1:1000), GSK-3β(CST, 1:1000), GAPDH (KangChen Bio-tech, 1:1000) at 4 °C overnight separately, and further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; keyGEN-BIO) for 1 h. Bands were visualized by Western Blotting Reagents (EMD Millipore, Billerica, MA, USA).
Gapdh
Manufactured by Kangchen Biotech
Sourced in China, United States
GAPDH is a key enzyme involved in the glycolysis pathway. It catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Nitrocellulose membrane, by Merck Group (4 mentions)
E cadherin, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
Vimentin, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Caspase 1 p20, by Adipogen (2 mentions)
Aanat, by Merck Group (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Nlrp3, by Adipogen (2 mentions)
Quantity one imaging software, by Bio-Rad (2 mentions)
Protease inhibitor, by Roche (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Nanog, by Abcam (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
37 protocols using gapdh
1
Histone H1 and Wnt Signaling Proteins
2
Bladder Cancer Cell Lines Culture Protocol
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Human BCa 5637, T24, 253J, 253J-BV cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in RPMI-1640 medium for 5637 cells or DMEM medium for T24, 253J and 253J-BV cells supplemented with 10% fetal bovine serum at 37 °C aired with 5% CO2. Highly lung metastatic T24-L subline cells expressing luciferase were generated as described previously and cultured in DMEM medium supplemented with 10% fetal bovine serum and 600 mg/l G418 at 37 °C aired with 5% CO2.20 (link) The MEK1/2 inhibitor U0126 and the antibiotic G418 were obtained from Sigma-Aldrich (St. Louis, MO, USA), and dissolved in DMSO and stored at −20 °C. The antibodies used were as follows: RASAL2 (rabbit, Abcam, Cambridge, UK), GAPDH (mouse, KangChen Bio-Tech, Shanghai, China), CD44 (mouse, Cell Signaling Technology, Beverly, MA, USA), SOX2 (rabbit, Cell Signaling Technology), E-cadherin (rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Vimentin (rabbit, Cell Signaling Technology), ZEB1 (rabbit, Cell Signaling Biotechnology), p-ERK1/2 (Thr202/Tyr204) and ERK1/2 (rabbit, Cell Signaling Technology), p-MEK1/2 (Ser217/221) and MEK1/2 (rabbit, Cell Signaling Technology).
3
Hippocampal Protein Expression Analysis
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Samples of hippocampus were homogenized on ice in PBS containing 1% protease inhibitor and 1% phosphatase inhibitor for Western-blotting. Proteins were obtained by centrifugation at 14000 rpm at 4°C for 15 min and quantified by Bradford assay (BioRad, USA). A 50 μg sample of each group was subjected to electrophoresis using 20% SDS at 80 V. The proteins were transferred to polyvinylidene fluoride membranes at 250 mA for 2 h. Antibodies for BDNF (1 : 1000, Santa Cruz Biotechnology, USA, number sc-546), TrkB (1 : 1000, Santa Cruz Biotechnology, USA, number sc-8316), CREB (1 : 2000, Cell Signaling Technology, USA, number 9197), pCREB (1 : 2000, Cell Signaling Technology, USA, number 9198), and GAPDH (1 : 10000, KangChen Bio-Tech, Shanghai, China) were applied overnight. Then membranes were incubated with horseradish peroxidase (HRP) secondary antibody (KangChen Bio-Tech, Shanghai, China). At last, the signal intensities of proteins were analyzed using Image J software.
4
Immunoblotting Analysis of Signaling Pathways
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Whole protein extracted from RAW264.7 macrophages were resolved by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels according to standard procedures. The samples were then transferred to polyvinylidene difluoride membranes (Bio-Rad) and incubated at 4 °C overnight with antibodies against phosphorylated IKK-α/β (1:500, Cell Signaling Technology), IKK-α (1:1000, Cell Signaling Technology), IKK-β (1:1000, Cell Signaling Technology), phosphorylated NF-κBp65 (1:500, Cell Signaling Technology), NF-κBp65 (1:1000, Cell Signaling Technology), phosphorylated I-κBα (1:1000, Cell Signaling Technology), I-κBα (1:1000, Cell Signaling Technology), PPAR-γ (1:500, Cell Signaling Technology), phosphorylated STAT6 (1:500, Abcam), STAT6 (1:1000, Abcam) and the endogenous control GAPDH (1:10000, KangChen Bio-tech). The blots were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000, KangChen Bio-tech) at room temperature for 1 h. Immunoreactive bands were detected with ECL Western blotting kit (Thermo Scientific Pierce) and exposed to films and developed. The density of the immunoblots was measured by Image J (National Institutes of Health) and normalized by GAPDH.
5
Protein Expression Analysis of Adipose Tissue
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Cultured cells or the subcutaneous and epididymal adipose tissues of mice were lysed in radioimmunoprecipitation buffer (Beyotime Biotechnology, P0013B) containing protease and phosphatase inhibitors. Lysates were centrifuged at 13,000 g for 30 min at 4 °C. Protein concentrations of the extracts were determined with a Pierce BCA Protein Assay Kit (Thermo, 23227). Membranes were incubated with GAPDH (KangChen Biotech, KC-5G4), HSP90 (Cell Signaling Technology, 4877), ACTIN (Cell Signaling Technology, 4967), Cathepsin B (CTSB) (Cell Signaling Technology, 31718), PLIN1 (Cell Signaling Technology, CST, 9349s), SQSTM1 (Cell Signaling Technology, 5114), and autophagy antibodies (Cell Signaling Technology, 4445), including BECN1, LC3A/B, ATG5, ATG16L1, ATG7, and ATG3 overnight at 4 °C. The membranes were incubated with an appropriate secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. ECL Prime Western blotting Detection Reagent (GE Healthcare, RPN2232) was used to visualize protein bands by electro-chemoluminescence (ImageQuant LAS4000, USA).
6
Western Blot Analysis of Signaling Pathways
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Western blot analysis was performed as previously described 19 (link),24 (link),25 (link). Cells were lysed in RIPA buffer supplemented with PMSF and protease inhibitor cocktail (Roche, Basel, Switzerland) on ice. Protein samples were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK) by standard methods. The membranes were immunoblotted with primary antibodies overnight at 4 °C. Antibodies against the following were used: p-p65 (1:1000; Cell Signaling Technology, Danvers, MA, USA), p65 (1:1000; Cell Signaling Technology), MAPK Family Antibody Sampler Kit (1:1000; Cell Signaling Technology), phospho-MAPK Family Antibody Sampler Kit (1:1000; Cell Signaling Technology), and GAPDH (1:5000; Kangchen Bio-tech, Shanghai, China). Horseradish peroxidase (HRP)-conjugated antibodies were used and visualized with the chemiluminescence substrate (Merck Millipore, Billerica, MA, USA).
7
Western Blot Analysis of HNF4α Protein
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The expression level of HNF4α protein was measured by western blotting as previously described [18 (link)]. The following antibodies were used: primary antibodies HNF4α (Cat. no. ab201460, 1 : 1000, Abcam) and GAPDH (Cat. no. KC-5G5, 1 : 8000, KangChen Bio-tech, Shanghai, China) and an HRP-conjugated secondary antibody (Cat. no. bs-0295G-HRP, 1 : 8000, Boster, Wuhan, China). Endogenous GAPDH was used as a loading control. The X-ray film of the internal reference control GAPDH and the target protein HNF4α band was photographed using a gel image scanning analysis system. The integrated optical density value of each band was measured by using the gray-scale analysis software Image-Pro Plus 6.0. The optical density ratio of the target protein/internal reference protein is the relative expression of the target protein.
8
Western Blotting for Colorectal Cancer Cell Analysis
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Colorectal cancer cells were dissolved in RIPA (Thermo Fisher Scientific, Waltham, MA, USA). Approximately 40 μg total protein was separated by 10% SDS‐PAGE and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 10% blocking solution (Roche, Basel‐Stadt, Switzerland) and incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibody for 1 h at room temperature. The bands were detected using the Odyssey Infrared Imaging System (LI‐COR Biosciences, Lincoln, NE, USA). The following primary antibodies were used: NUBPL (SAB1408017, Sigma‐Aldrich), E‐cadherin, α‐catenin, N‐cadherin, vimentin, fibronectin, α‐smooth muscle actin (α‐SMA) (all from Proteintech, Rosemont, IL, USA), ERK, phospho‐ERK (both from Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Kangchen Bio‐tech, Shanghai, China).
9
Western Blot Analysis of Protein Expression
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Total protein was extracted by radioimmunoprecipitation assay buffer (Beyotime, Shanghai, People’s Republic of China) adding phenylmethylsulfonyl fluoride (Beyotime). Protein extracts (50–100 µg) in each condition were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Beyotime) and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk, the membranes were incubated with the following primary antibodies at 4°C overnight: PF4V1 (1:2,000; Thermo Fisher Scientific, Waltham, MA, USA), ERK (1:1,000; Abcam, Cambridge, UK), p-ERK (1:1,000; Abcam), AKT (1:1,000; Abcam), p-AKT (1:2,000; Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:1,000; Abcam), N-cadherin (1:2,000; Cell Signaling Technology), SRC-1 (1:2,500; Cell Signaling Technology), Snail (1:2,000; Cell Signaling Technology), Slug (1:2,000; Cell Signaling Technology), or GAPDH (1:10,000; KangChen Biotech, Shanghai, People’s Republic of China). The enhanced chemiluminescence reagent (Thermo Fisher Scientific) was used to detect the intensity of protein bands.
10
Western Blot Protein Detection Protocol
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Cells were washed with phosphate-buffered saline (PBS) and lysed in 500 μl RIPA buffer with 5 μl protease inhibitor cocktail (100X, Merck, USA) on ice for 15 min, then centrifugation at 13 000 g for 10 min. The proteins in 20 μl supernatant were separated with 12% SDS-PAGE and transferred onto a nitrocellulose membrane. Then the membrane was incubated with rabbit anti-human primary antibody PAK4 (1:500–1:1000, Abcam, UK), mouse Anti-Flag Tag (1:1000–1:10 000, Proteintech, USA) and mouse anti-human primary antibody GAPDH (1:5000, KangChen Bio-tech Inc. China) at 4 °C overnight, respectively. Next, it was incubated with a horseradish peroxidase-labeled (HRP-labeled) goat anti-rabbit or anti-mouse secondary antibody (1:2000, Jackson Immunotech, UK) at room temperature for 1 h. The membrane was developed using enhanced chemiluminescence (ECL, Millipore, Germany) for testing.
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