Samples were homogenized using RIPA buffer (Beyotime, Shanghai, China) with Protease and Phosphatase Inhibitor (Thermo Fisher) or Nuclear-Cytosol Extraction Kit (Fdbio science, Hangzhou, China). Proteins were separated in SDS-PAGE and then transferred to the PVDF membranes (Merck Millipore). After blocking with 5% bovine serum albumin, the membranes were incubated with antibodies raised against Estrogen Receptor alpha (1:1000, ab32063, Abcam, USA), PCNA (Proteintech, USA), NLRP3 (1:1000, ab263899, Abcam), cleaved caspase-1 (1:1000, 89332, Cell Signaling Technology), pro-caspase-1 (1:1000, ab179515, Abcam), IL-1β (1:1000, 12242, Cell Signaling Technology), ASC (1:1000, 67824, Cell Signaling Technology), and GAPDH (1:1000, Mab5465, MultiSciences, Hangzhou, China). After incubation with HRP-conjugated secondary antibody against rabbit IgG (1:10000, ab97051, Abcam) or against mouse IgG (1:10000, ab97023, Abcam), the membranes were detected using the ECL detection kit (Biological Industries) and imaged on the ImageQuant LAS 4000 Mini Biomolecular Imager (GE Healthcare Life Sciences, USA). The detected protein expression was quantified on band volume by Image J and normalized over GAPDH mean band intensity.
Mab5465
Manufactured by MultiSciences Biotech
Sourced in China, United States
Mab5465 is a laboratory instrument designed for the detection and quantification of specific proteins or molecules in a sample. It utilizes monoclonal antibody technology to perform highly sensitive and selective analyses.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (2 mentions)
Ab179515, by Abcam (1 mentions)
Ab97023, by Abcam (1 mentions)
Ab32063, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 mini biomolecular imager, by GE Healthcare (1 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)
Nuclear cytosol extraction kit, by Fdbio (1 mentions)
Ecl detection kit, by Sartorius (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab263899, by Abcam (1 mentions)
Cyp2e1, by Abcam (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Gapdh, by MultiSciences Biotech (1 mentions)
Goat anti rabbit igg, by MultiSciences Biotech (1 mentions)
Chemi xr 5, by Syngene (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
3 protocols using mab5465
1
Protein Expression Analysis in Cell Samples
2
Quantification of Protein Expression in Tissues
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Lung and liver tissues and L-02 cells were lysed in RIPA buffer, and a BCA kit (Beyotime Biotechnology) was used to quantify the level of protein in a sample. Protein bands were separated by polyacrylamide gels electrophoresis (SDS-PAGE, 8–14%) and then repositioned on PVDF membranes. After appropriate blockade, PVDF membranes were incubated with the following primary antibodies: NF-κB p65 (1:1,000) (Cell Signaling Technology, Boston, MA, USA); PXR; CYP3A1, CYP1A2, and CYP2E1 (1:1,000) (Abcam, Cambridge, MA, USA); GAPDH (1:1,000) (MAB5465, MultiSciences, Hangzhou, China); and goat antirabbit IgG (1:5,000) (70-GAR007, MultiSciences), then incubated at room temperature with secondary antibodies for 1 h. ECL chemicals (Beyotime Biotechnology) were used to examine the target proteins. An image analyzer system (ChemiDoc XRS+; Bio-Rad, Hercules, CA, USA) was used to quantify the results.
3
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).
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