For extraction of the total protein, left ventricular heart tissues from rats were homogenized in commercial RIPA buffer (Boster, Wuhan, China) added with protease inhibitor and phosphatase inhibitor (MedChemExpress, America). Then, the supernatants were obtained after centrifugation at 12000×g at 4°C for 20 min. The protein concentration was detected using a bicinchoninic acid assay (Boster, Wuhan, China). Denatured proteins (40 μg/lane) were loaded on and separated by 8-10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels and transferred onto polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% BSA at room temperature for 1 h, which subsequently were incubated with primary antibodies overnight at 4°C. After washing with TBS-T, the membranes were probed with peroxidase-conjugated secondary antibodies (Boster, Wuhan, China) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit following the manufacturer's instructions (New Cell & Molecular Biotech, Suzhou, China). Protein expression contents were analyzed by ImageJ software, and the level of β-actin or GAPDH was used as an internal control. The primary and secondary antibodies used in this study were listed in Supplementary Table 1 .
Protease inhibitor and phosphatase inhibitor
Manufactured by MedChemExpress
Sourced in China
Protease inhibitor and phosphatase inhibitor are lab equipment used to inhibit the activity of proteases and phosphatases, respectively. Proteases are enzymes that break down proteins, while phosphatases are enzymes that remove phosphate groups from other molecules. These inhibitors are commonly used in various research applications to prevent unwanted protein degradation or dephosphorylation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid assay, by Boster Bio (1 mentions)
Ripa buffer, by Boster Bio (1 mentions)
Peroxidase conjugated secondary antibody, by Boster Bio (1 mentions)
Enhanced chemiluminescence reagent, by Beyotime (1 mentions)
Ab92700, by Abcam (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab32536, by Abcam (1 mentions)
Ab263899, by Abcam (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
Ab283818, by Abcam (1 mentions)
Magnetic beads, by Selleck Chemicals (1 mentions)
Wash buffer, by Thermo Fisher Scientific (1 mentions)
Ip lysate, by Thermo Fisher Scientific (1 mentions)
Ripa cell lysis solution, by Solarbio (1 mentions)
Ecl luminescent solution, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
3 protocols using protease inhibitor and phosphatase inhibitor
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis in Kidney
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Total protein in kidney tissues was lysed in RIPA lysis buffer (Beyotime) containing protease inhibitor and phosphatase inhibitor (MedChemExpress). After being quantified with a BCA Protein Assay kit (Beyotime), protein samples (30 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% skimmed milk in PBS solution with 0.05% Tween (PBST; Thermo Fisher Scientific) for 1 h at room temperature and incubated with rabbit anti-p-NF-κB p65 antibodies (ab76302, 1:1000; Abcam), rabbit anti-IκB antibodies (ab92700, 1:1000; Abcam), mouse anti-β-actin antibodies (ab8226, 1:1000; Abcam), rabbit anti-NF-κB p65 antibodies (ab32536, 1:2000; Abcam), rabbit anti-histone H3 antibodies (ab1791, 1:1000; Abcam), rabbit anti-N--LRP3 antibodies (ab263899, 1:1000; Abcam), rabbit anti-cleaved caspase-1 antibodies (#89332, 1:1000; Cell Signaling Technology, Shanghai, China), and rabbit anti-IL-1β antibodies (ab283818, 1:1000; Abcam) at 4℃ overnight. Subsequently, the membranes were washed with PBST and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoblots were developed using enhanced chemiluminescence reagent (Beyotime). Densitometric analysis was performed using ImageJ software (Bio--Rad, Hercules, CA, USA).
3
Western Blot and Immunoprecipitation Analysis
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Cells were first lysed with RIPA cell lysis solution (Solarbio, China) containing protease inhibitor and phosphatase inhibitor (MedChemExpress, USA), the lysate was separated by SDS-PAGE gel and transferred to PVDF membranes (Millipore, USA), then incubated with proteins successively according to the instructions of primary and secondary antibodies. Finally, exposure was performed using the C300 system (Azure Biosystems, USA) in the presence of an ECL luminescent solution (Millipore, USA). For immunoprecipitation, the starting steps were as described above but involved the use of IP lysate (Thermo Fisher Scientific, USA). A portion of the protein solution was taken to directly detect the corresponding protein expression, while the rest of the solution was added to the primary antibody and magnetic beads (Bimake, USA), respectively, according to the manufacturer's instructions. The beads were washed with wash buffer (Thermo Fisher Scientific, USA) and heated to 100°C for 5 min using protein loading buffer (Abclonal, China). Immunoblotting was then performed as described above.
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