Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis reagent (Thermo Fisher). The protein concentration was determined using a Bradford protein assay (Sigma-Aldrich). A 50-μg sample was separated on a 10% denaturing polyacrylamide gel (with 5% polyacrylamide stacking gel) and transferred electrophoretically onto a nitrocellulose membrane. After blocking with 5% nonfat dry milk in Tris-buffered saline plus Tween (TBST), the membranes were incubated with primary antibodies and then incubated with HRP-conjugated secondary antibody (Thermo Fisher). Bands were visualized by chemiluminescence reaction using an enhanced chemiluminescence detection system (Thermo Fisher) and captured using Bio-Rad Imaging Systems (Bio-Rad).
Ripa lysis reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RIPA lysis reagent is a commonly used buffer solution designed for cell lysis and protein extraction. It contains a combination of detergents, salts, and other components that facilitate the efficient disruption of cell membranes and the solubilization of cellular proteins. This reagent is suitable for a wide range of applications, including Western blotting, immunoprecipitation, and other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bradford protein assay, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Imaging systems, by Bio-Rad (1 mentions)
Ab188570, by Abcam (1 mentions)
Af1785, by R&D Systems (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Foxm1, by Cell Signaling Technology (1 mentions)
P jak2, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence ecl detection system, by Thermo Fisher Scientific (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
5 protocols using ripa lysis reagent
1
Western Blot Protein Detection Protocol
2
Regulation of COL2A1 and MMP16 in Chondrocytes
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The protein expression of COL2A1 and MMP16 were examined by western blot analysis. The chondrocytes were treated with miR-193b-3p mimics, mimics-NC, MMP16 expression or DMEM. The specimens were washed twice using PBS and lysed using a RIPA lysis reagent (Thermo Fisher Scientific, Rockford, Illinois, USA). The protein concentrations were determined by bicinchoninic acid (BCA) assay (Pierce Biotechnology, Waltham, Massachusetts, USA). The samples were incubated overnight with primary antibodies including rabbit anti human COL2A1 (Abcam, ab188570, 1:1000 dilution) and goat anti human MMP16 (R&D, AF1785, 1:10000 dilution) at 4°C. The membrane for each antibody was then visualized with horseradish peroxidase (HRP) conjugated secondary antibodies (goat anti-rabbit and donkey anti-goat, Daixuan Bio, 1:500 dilution). The intensities of the protein bands, representative of protein levels, were determined using Quantity One Image software. β-actin was used to normalize target proteins. All experiments were performed in triplicate.
3
Quantitative Western Blot Analysis
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Cell proteins were extracted using RIPA lysis reagent (Thermo Fisher Scientific, Inc.) with protease and phosphatase inhibitors. The total protein concentration was determined using a Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Inc.). Proteins were stored at −80°C and were then separated by SDS-PAGE on a 12% gel at a voltage of 120 V for 2 h. Subsequently, proteins were transferred to PVDF membranes, which were blocked with 5% BSA (Merck & Co., Inc.) for 2 h, incubated with primary antibodies for 4 h at room temperature and incubated with a secondary antibody for 2 h at room temperature. Finally, electrochemiluminescence was captured by a GE LAS-600 Imaging system with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.). The gray value of bands was semi-quantified using ImageJ software V1.5.0 (National Institutes of Health). Details of the antibodies involved in this study are shown in Table I .
4
CDKN3 Protein Expression Analysis
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CDKN3 protein expression was determined using western blotting in the three cell lines transfected with the specific CDKN3 or scrambled siRNAs. Proteins were obtained in radioimmunoprecipitation assay (RIPA) lysis reagent (Cat: 89900, Life Technologies). The protein content of each lysate was determined using a Bradford protein assay (Cat: B6916, Sigma-Aldrich, St Louis, MO, USA) with bovine serum albumin (BSA) as a standard. A 50-μg sample of each lysate was resolved on 12% denaturing polyacrylamide gels (with 5% polyacrylamide stacking gel) and transferred electrophoretically onto a nitrocellulose membrane. After blocking with 5% nonfat dry milk in Tris-buffered saline plus Tween (TBST), the membrane was incubated with a rabbit polyclonal antihuman CDKN3 antibody (sc-475 Santa Cruz Biotechnology, Inc.) and a mouse monoclonal antihuman actin antibody (the monoclonal anti-actin antibody was kindly donated by Manuel Hernámdez, Ph.D, CINVESTAV) overnight at 4°C. The membrane was then incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies (GE Healthcare Bio-Sciences) for 1 h at room temperature. After washing with TBST, the immunoreactive proteins were developed using the enhanced chemiluminescence kit (GE Healthcare Bio-Sciences).
5
Western Blot Analysis of Signaling Proteins
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Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis reagent (Life Technologies, Carlsbad, CA, USA). Protein lysates were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat dry milk in Tris-buffered saline plus Tween (TBST), the membranes were incubated with primary antibodies [β-actin, 1:1,000 (Abcam); BTF3, 1:1,000; FOXM1, 1:1,000; STAT3, 1:1,000; p-STAT3, 1:1,000; JAK2, 1:1,000; p-JAK2, 1:1,000 (all from Cell Signaling Technology)] and then incubated with HRP-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA). Blots were detected using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific) and captured using Bio-Rad Imaging Systems (Bio-Rad, Hercules, CA, USA).
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