Western blot was performed following standard protocols, detailed as follows: cells were harvested and lysed using RIPA buffer (Beyotime, Shanghai, China). Protein extracts were subjected to SDS-PAGE analysis. The membranes were blocked with 5% BSA buffer followed by antibody hybridization and then visualized in G-BOX gel imaging system (Chemi XR 5, Syngene, Cambridge, UK).
Chemi xr 5
Manufactured by Syngene
Sourced in United Kingdom
The Chemi XR 5 is a chemiluminescence imaging system designed for the detection and analysis of proteins and nucleic acids. It features a high-resolution CCD camera, selectable emission filters, and software for image capture and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Mab5465, by MultiSciences Biotech (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Blood tissue cell genome extraction kit, by Tiangen Biotech (1 mentions)
Ab1191, by Abcam (1 mentions)
Pepstatin, by Beyotime (1 mentions)
Nb100 105, by Novus Biologicals (1 mentions)
Ab4729, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Nb100 122, by Novus Biologicals (1 mentions)
Mem per plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Leupeptin, by Beyotime (1 mentions)
4 protocols using chemi xr 5
1
Western Blot Analysis of Protein Extracts
2
Protein Extraction and Western Blot Analysis
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Protein was extracted with RIPA lysate (Beyotime Biotechnology, P0013 B) and the protein concentration was determined by BCA kit (Beyotime Biotechnology, P0010), and then the equal amounts of protein extracts were subjected to SDS-PAGE analysis and then and then transferred to polyvinylidene fluoride (PVDF) membranes. The concentrations of separation gel and concentrated gel were 10% and 5%, respectively. The PVDF membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4°C, and the second antibody was incubated for 2 h at room temperature. Finally, the PVDF membranes were visualized in G-BOX gel imaging system (Chemi XR 5, Syngene). Antibodies were used as follows: anti-TET1 antibody (Gene Tex, GT465), anti-E-cadherin antibody (abcam, ab133597), anti-GAPDH antibody (MULTI SCIENCES, Mab5465), and anti-beta-actin antibody (MULTI SCIENCES, ab-40009-100).
3
DNA Extraction and 5hmC Detection
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Tissue and cellular DNA extraction was performed using the Tiangen Blood/tissue/cell genome extraction kit (TIANGEN, DP304) and the concentration was detected by NanoDrop. The extracted DNA was diluted to the same concentration with ddH2O, and 4 M NaOH solution was added to a final concentration of 0.4 M, then maintained in 95°C water bath 10 min after thoroughly mixed. 1 μL DNA sample was spotted on a nylon membrane, and then irradiated for 15 min for cross-linking by UV lamp. The cross-linked nylon membrane was blocked in 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies overnight at 4°C, and the second antibody was incubated for 2 h at room temperature. Finally, the nylon membrane was visualized in G-BOX gel imaging system (Chemi XR 5, Syngene). anti-5hmC antibody was purchased from active motif.
4
Membrane Protein Expression Analysis
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Cells were harvested and lysed using RIPA buffer (Beyotime) supplemented with PMSF (Beyotime), leupeptin (Beyotime) and pepstatin (Beyotime). The membrane proteins were extracted by Mem-PER™ Plus Membrane Protein Extraction kit (Thermo Fisher, USA). The protein concentrations of lysate were determined by bicinchoninic acid (BCA) assay. Protein extracvts were subjected to SDS-PAGE analysis and transferred to polyvinylidene difluoride (PVDF) membrane after electrophoresis. The membranes were blocked with 5% BSA buffer followed by antibody hybridization and then visualized in G-BOX gel imaging system (Chemi XR 5, Syngene). Antibodies and dilution ratio in this study include anti-OCT2 (HPA008567, Sigma-Aldrich) at 1:200 dilution, anti-GAPDH (KC-5G4, Kangchen) at 1:5000 dilution, anti-ATP2A2 (AB54032, Sangon Biotech) at 1:5000 dilution, anti-ENT1 (11337-1-AP, proteintech) at 1:1000 dilution, anti-HIF-1α (610958, BD Biosciences), (NB100-105, Novus) at 1:800 dilution, anti-HIF-2α (NB100-122, Novus) at 1:1000 dilution, anti-H3K27ac (ab4729, Abcam) at 1:3000 dilution, anti-H3K18Ac (ab1191, Abcam) at 1:3000 dilution and anti-H3 (ab1791, Abcam) at 1:3000 dilution.
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