Cells were directly lysed with RIPA containing protease and phosphatase inhibitors (Roche Applied Science) and proteins were separated by 10% SDS-PAGE after denaturation. Immunoblot analysis was performed by initial transfer of proteins onto polyvinylidenefluoride membranes using Mini Trans-Blot (Bio-Rad Laboratories, Richmond, VA, USA) and followed by a blocking step with 5% nonfat dried milk plus 0.1% Tween 20 for 2 hours at room temperature and exposed to primary antibodies diluted 1,000-fold that recognized Slug, osteocalcin, osteopontin, Runx2, VE-cadherin, occludin, N-cadherin, vimentin or β-actin overnight at 4 °C and subsequently washed. The blots were then incubated with a secondary Ab conjugated with Horse Radish Peroxidase diluted 5,000-fold for 1 hour at room temperature. Signals were detected by FluorChem E system (Alpha Innotech Corp, Santa Clara, CA, USA).
Ripa containing protease and phosphatase inhibitors
Manufactured by Roche
Sourced in Switzerland, United States
RIPA (Radioimmunoprecipitation Assay) buffer containing protease and phosphatase inhibitors is a solution used for the extraction and solubilization of proteins from cells and tissues. It facilitates the lysis of cells and the subsequent isolation of proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mini trans blot, by Bio-Rad (2 mentions)
Enhanced chemiluminescence reagent kit, by Cytiva (1 mentions)
Gel image analysis system, by Bio-Rad (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions)
Azure c400, by Azure Biosystems (1 mentions)
Azurespot analysis software, by Azure Biosystems (1 mentions)
4 protocols using ripa containing protease and phosphatase inhibitors
1
Western Blot Analysis of Cell Lysates
2
Western Blot Analysis of Kidney Proteins
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Kidney tissues or HK-2 cells were directly lysed with RIPA containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Total proteins were separated by 10% SDS-PAGE after denaturation and transferred onto nitrocellulose membranes. After blocking, the membranes were incubated with rabbit anti-mouse monoclonal antibodies against SIRT3, NLRP3, ASC, caspase-1, Bax, Bcl-2, caspase-3 or β-actin (1:400, Cell Signaling, Denvers, USA) overnight at 4 °C with shaking, and then incubated with the respective peroxidase-conjugated goat anti-rabbit IgG (1:2000, Santa Cruz, CA, USA) for 1 h at room temperature. The membranes were washed three times, and then detected using an enhanced chemiluminescence reagent kit (Amersham Life Science, Cleveland, USA). The gray values were determined by a gel image analysis system (Bio-Rad, Hercules, USA) and normalized to β-actin.
3
Western Blot Analysis of EZH2 in Purified CD3+CD25+ T Cells
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CD3+CD25+ T cells were sorted out from MNCs by flow cytometry. The purity of sorted cells in this study was consistently more than 99 %. Then, cells were directly lysed with RIPA containing protease and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN, USA) and proteins were separated by 10 % SDS-PAGE after denaturation. Immunoblot analysis was performed by initial transfer of proteins onto polyvinylidenefluoride membranes using Mini Trans-Blot (Bio-Rad Laboratories, Richmond, VA, USA) and followed by a blocking step with 5 % nonfat dried milk plus 0.1 % Tween 20 for 2 h at room temperature and exposed to primary antibodies diluted 1000-fold that recognized EZH2 and actin overnight at 4 °C and subsequently washed. The blots were then incubated with a secondary antibody conjugated with Horse Radish Peroxidase diluted 5000-fold for 1 h at room temperature. Signals were detected by FluorChem E system (Alpha Innotech Corp, Santa Clara, CA, USA).
4
Pneumococcal Infection Response in PM2.5-Treated Macrophages
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RAW264.7 cells (2 × 106) were untreated or treated with PM2.5 (20 μg/ml) for 24 h followed by infection with pneumococcus (MOI = 10) for an additional 6 h. Cells were washed and lysed with 100 μl RIPA containing protease and phosphatase inhibitors (Roche, Indianapolis, IN), and then subjected to western blot assay. The samples were resolved by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked by 5% skim milk and incubated with the primary antibodies followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Millipore). The proteins of interests were detected using ECL Western Blotting Detection Reagent (BIOMAN, Taipei, Taiwan) and analyzed by Azure C400 (Azure Biosystems, Dublin, CA) and AzureSpot Analysis Software (Azure Biosystems) [27 (link)]. To determine the intensities of western blot bands, Un-Scan-It v6.1 software (Orem, UT, USA) was used. Identical areas surrounding each band were cropped, and protein expression levels were converted into pixel densities. Each area value was normalized to the β-actin density in the same lane on the gel, and then divided by the normalized density in the mock-control. Fold change represents protein expression level relative to the mock-control.
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