The protein was extracted with RIPA Lysis buffer (Boster, AR0102-100, Wuhan, China) and quantified by ultrastructure nucleic acid protein tester (Analytik Jena, Jena, Germany). Equal amounts of protein were separated by SDS-PAGE, immunoblotted with antibodies IRE1α (Proteintech, 27528-1-AP, Wuhan, China), anti-TRAF2 antibody (Abcam, ab126758, Cambridge, UK), anti-IKK-β antibody (Abcam, ab124957, Cambridge, UK), IκB-α antibody (CST, #4812, Boston, MA, USA), NF-κBp65 antibody (CST, #8242, Boston, US), ASK1 antibody (Proteintech, 67072-1-Ig, Wuhan, China), anti-JNK1 antibody (Abcam, ab199380, Cambridge, UK), and GAPDH antibody (Proteintech, 60004-1-IG, Wuhan, China). Antibody binding was detected using horseradish peroxidase-conjugated anti-rabbit IgG/anti-mouse IgG antibody (Proteintech, SA00001-1/SA00001-2, Wuhan, China) and visualized with Chemiluminescence Solution (Abbkine, BMU102-CN, Wuhan, China).
Bmu102 cn
Manufactured by Abbkine
Sourced in China
The BMU102-CN is a laboratory instrument designed for the analysis and detection of biomolecules. It is a versatile device that can be used in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Boster Bio (1 mentions)
Ab126758, by Abcam (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Protease inhibitor, by Targetmol (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Super nuclease, by Beyotime (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
S8830, by Merck Group (1 mentions)
4 protocols using bmu102 cn
1
Western Blot Analysis of Signaling Proteins
2
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) for 30 min and centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatants were collected. The protein concentration was determined using the BCA method (Thermo Scientific, 23222, USA). The proteins were separated by SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Merck Millipore, ISEQ00010, USA). The PVDF membrane was incubated for 1 h in 5% skim milk powder at room temperature and then incubated with the corresponding anti-antibody overnight at 4°C. After washing thrice for 10 min with PBST, the membrane was incubated with the secondary antibody at 37°C for 1 h. The protein bands were visualized using enhanced chemiluminescence reagents (Abbkine, Wuhan, China, BMU102-CN). The ChemiDoc imaging system (Bio–Rad, USA) was used to capture the images and quantify the intensity of the protein fragments.
3
Protein Extraction and Western Blotting
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Twenty milligrams of brain tissue from the temporal lobe of the injured hemisphere were collected and cut into pieces. Cells were collected directly and centrifuged. Cultured cells or brain tissue were lysed on ice with a mixture of RIPA (#89,901, Thermofisher, USA), 1% protease inhibitor (#C0001, TargetMol, USA), and 0.1% super nuclease (#D7121-5KU, Beyotime). Protein quantification was performed using the Dual Chondroitin Protein Assay Kit (#P0009, Beyotime). The same mass of proteins was loaded onto sodium dodecyl polyacrylamide gels (#PG111, Epizyme, China) before transferring to polyvinylidene fluoride membranes (#IPVH00010, Sigma, USA). The membranes were blocked with 5% skim milk powder for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature. We used a standard electrochemiluminescence substrate (#BMU102-CN, Abbkine, USA), incubated the blots with the working solution for 1-5 min, and analyzed the bands using ImageJ. Antibodies used in WB are listed in Table 1.
4
Western Blot Analysis of Protein Lysates
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Cells or xenograft tumors were lysed using RIPA (catalog V900854-100ML, VETEC) with 1 mM PMSF, and protease inhibitors (catalog S8830, Sigma‒Aldrich). Protein concentrations were quantified using the BCA protein test package (catalog P0010, Beyotime). After the proteins were separated, they were transferred to a PVDF membrane (catalog IPVH00010, Millipore), blocked with 5% nonfat milk, and incubated with the corresponding antibodies. On the second day, the bands were incubated with the corresponding secondary antibodies. HRP chemiluminescent substrates were used to visualize the immunoreactive bands (catalog BMU102-CN, Abbkine). The bands were quantified from the western blot results using Image Lab. The antibodies used in this study are listed in Supplementary Table 2 .
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