Seventy-two hours after transfection, total protein was extracted from each group with RIPA lysates (Epizyme, China) containing protease inhibitors (Epizyme, China) (1 : 100) and nucleases (Haigene, China) (1 : 100) and quantified with BCA Protein Assay Kit (Cwbio, China). After each lane was loaded with 20 μg protein by SDS-PAGE gel electrophoresis, the protein ladders were transferred to a PVDF membrane (Milipore, USA). And then, the membrane was blocked at room temperature for 5 minutes with a fast blocking solution (Epizyme, China) (1 : 100) and incubated with rabbit anti-monkey IκBα monoclonal antibody (CST, USA) (1 : 5000) and rabbit anti-monkey ß-actin monoclonal antibody (CST, USA) (1 : 10000) overnight at 4°C. Then HRP-labeled goat anti-rabbit IgG secondary antibody (CST, USA) (1 : 5000) was added and incubated for 1 hour at room temperature. Specific signals were visualized with Immobilon Western chemiluminescence HRP Substrate (Milipore, USA) and acquired with ChemiDocTM Touch Imaging System (Bio-Rad, USA). The gray value of each band and the relative expression of IκBα protein were calculated and analyzed by Gel-Pro analyzer software version 4.
Ripa lysate
Manufactured by Ipsen
Sourced in China
RIPA lysate is a cell lysis buffer used in protein extraction and purification procedures. It is designed to effectively solubilize and extract proteins from cells and tissues. The buffer contains a combination of detergents, salts, and buffers that help to disrupt cell membranes and release proteins into the solution.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Ipsen (3 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by CWBIO (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Immobilon western chemiluminescence hrp substrate, by Merck Group (1 mentions)
Chemiluminescence reagent kit, by Beyotime (1 mentions)
β actin, by Bioss Antibodies (1 mentions)
Loading buffer, by Solarbio (1 mentions)
Bca kit, by Biosharp (1 mentions)
Bca protein quantification kit, by Ipsen (1 mentions)
Vcam 1, by Proteintech (1 mentions)
Pvdf membrane, by Proteintech (1 mentions)
Hrp labeled goat anti mouse secondary antibody, by Proteintech (1 mentions)
Bca kit, by GenStar (1 mentions)
Icam 1, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
5 protocols using ripa lysate
1
Western Blot Analysis of IκBα Protein
2
Protein expression analysis in MCM and MTM cells
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Total protein was extracted from MCM and MTM cells with RIPA lysates (Epizyme, China) containing protease inhibitors (1:100; Epizyme, China) and nucleases (1:100; Hai-gene, China). The protein concentration of each sample was measured with a BCA Protein Assay Kit (Cwbio, China). After electrophoresis and transfer, the proteins were transferred to a 0.45-μm polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Then, the membranes were blocked at room temperature (RT) for 15 minutes with a fast blocking solution (1:5; Epizyme, China) and incubated with selected primary antibodies at 4°C overnight. The membranes were washed in Tris-buffered saline containing 0.1% Tween 20 (1X TBST) and incubated with secondary antibodies for 1 hour at RT. Finally, the membranes were washed and imaged using the ChemiDocTM Touch Imaging System (Bio–Rad, USA). The following primary antibodies were used: rabbit anti-IκBα (1:5000; CST, USA), rabbit anti-NF-κB P65 (1:1000; CST, USA), rabbit anti-phospho-NF-κB P65 (1:1000; CST, USA), mouse anti-MMP2 (1:500; Invitrogen, USA), and rabbit anti-β-actin (1:10000; CST, USA). The secondary antibodies were HRP-labeled goat anti-rabbit IgG (1:5000; CST, USA) and HRP-labeled goat anti-mouse IgG (1:5000; CST, USA). The experiment was repeated three times independently. The gray value of each band was calculated and analyzed using ImageJ software.
3
Western Blot Analysis of NUP205 in Brain Tissue
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An appropriate amount of brain tissue was homogenized with RIPA lysate (EpiZyme, China) and a protease inhibitor (EpiZyme, China). The brain tissue was then split on ice for 30 min, and the protein supernatant was centrifuged at 12000 rpm for 30 min at 4 °C. The protein concentration was detected using a BCA kit (Biosharp, China). Briefly, the protein was boiled at 100 °C for 10 min in 4× loading buffer (Solarbio, China), separated by SDS-PAGE electrophoresis, and then transferred to a PVDF membrane (Bio-Rad, USA). After the membrane was sealed with 5% evaporated milk, NUP205 (1:1000; Proteintech, China) and β-actin (1:1000; Bioss, China) primary antibodies were added overnight at 4 °C. Then, the membrane was incubated in goat anti-rabbit IgG H&L antibody (1:2000; Bioss, China) at 37 °C for 1 h. Finally, the protein blots were developed with a chemiluminescence reagent kit (Beyotime Biotechnology), and ImagePro-Plus software (version 6.0) was used for the quantitative analysis.
4
Western Blot Protein Analysis
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To begin with, cells were lysed using RIPA lysate (Epizyme, Shanghai, China) while being kept on ice. The protein concentration was detected using the BCA protein quantification kit (Epizyme, Shanghai, China). Next, gels were prepared and 20 μg of protein was loaded for electrophoresis using the PAGE Rapid Gel Preparation Kit (Epizyme, Shanghai, China). The protein gel was then transferred to a PVDF membrane. After the membrane transfer, the PVDF membrane was soaked in TBST solution containing 5% skimmed milk for 1 h at room temperature. The primary antibody was added and incubated overnight at 4 °C in the refrigerator. The next day, the membranes were incubated with HRP-labeled secondary antibody at room temperature for 1 h. Finally, the proteins on the membrane were visualized using an ECL ultrasensitive luminescent solution (Kemix, Zhengzhou, China).
5
Protein Expression Analysis in Transfected Cells
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The transfected cells were added to RIPA lysate and protease inhibitor to extract total protein (EpiZyme, Shanghai, China). After lysis on ice for 30 min, the proteins were centrifuged at 12000 rpm for 15 min at 4°C. The loading volume of each sample was measured using a BCA kit (GenStar, Beijing, China). Identical masses of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to PVDF membranes (Bio-Rad, UK) and sealed with skim milk powder. PVDF membranes were incubated overnight at 4°C with primary antibodies against VCAM-1 (1:500, Proteintech, USA), ICAM-1 (1:2500, Proteintech, USA), CDH2 (1:1500, Proteintech, USA), and GAPDH (1:10000, Proteintech, USA). Finally, the HRP-labeled goat anti-mouse secondary antibody (1:5000, Proteintech, USA) was incubated with the filter membrane for 1 h. GNG12 protein expression levels were detected using an imager and a chemiluminescent substrate (ECL) kit (Thermo Fisher Scientific, USA).
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