Frozen endometrial tissues or cells were lysed in 300ul lysis buffer containing a protease inhibitor (EpiZyme, Shanghai, China). After the protein concentration was determined by BSA method, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, and the protein was transferred to PVDF membrane. After TBST containing 5% fat-Free milk was blocked at room temperature for 2 h, the primary antibody was incubated, PKR1 antibody (Affinity, orb162427) 1: 500, PK2 antibody (Abcam, ab76747) 1: 200, MMP9 (N-terminal) Polyclonal antibody (Sanying,10,375-2-AP) 1: 100, IGFBP1 Polyclonal antibody (Sanying, 13,981-1-AP) 1:1000, PRL antibody (Abcam, ab188229) 1:2000, GAPDH Polyclonal antibody (Sanying, 10,494-1-AP) 1: 10000 overnight at 4 C. Next day, the PVDF membranes were washed by TBST 5-6 times* 5 min / per; were added horseradish peroxidase conjugated secondary antibody immunoglobulin G (IgG), were incubated at room temperature for 2 h, the membranes were finally revealed using electro-chemiluminescence (ECL) substrate and exposed to films.
Protease inhibitor
Manufactured by Ipsen
Sourced in China
Protease inhibitors are a class of laboratory equipment used to inhibit the activity of proteases, which are enzymes that break down proteins. They are commonly used in biological research, pharmaceutical development, and diagnostic applications to maintain the integrity of protein samples by preventing unwanted protein degradation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ripa lysate, by Ipsen (3 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Ipsen (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Bca protein assay kit, by CWBIO (2 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)
Pvdf membrane, by Bio-Rad (1 mentions)
Gapdh, by Proteintech (1 mentions)
Bca protein assay kit, by Ipsen (1 mentions)
Ecl chemiluminescence kit, by Ipsen (1 mentions)
Anti gapdh 10494 1 ap, by Proteintech (1 mentions)
Immobilon western chemiluminescence hrp substrate, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti gapdh, by Bioworld Technology (1 mentions)
F7425, by Abcam (1 mentions)
7 protocols using protease inhibitor
1
Protein Expression Analysis of Endometrial Tissues
2
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).
3
Protein Quantification and Western Blot Analysis
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Cells were collected 72 h after transfection and lysed using RIPA lysis buffer (Epizyme, Shanghai, China) and a protease inhibitor (Sollerbauer, Beijing, China). Protein concentrations were measured using the BCA Protein Assay Kit (Epizyme) and adjusted to equal concentrations. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, USA). The membranes were blocked with 5% skimmed milk for 2h at room temperature and incubated overnight at 4 °C with anti-RBM39 (21339-1-AP, 1:1,000, Proteintech, Wuhan, China) and anti-GAPDH (10494-1-AP, 1:5,000, Proteintech) primary antibodies. The immunoblots were detected using ECL chemiluminescence kit (Epizyme).
4
Western Blot Analysis of IκBα Protein
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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.
5
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.
6
Quantitative Protein Expression Analysis
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HEK293T cells were lysed in radioimmunoprecipitation buffer (EpiZyme, PC103) with 1 mM PMSF solution and a mixture of protease inhibitors (Roche), at about 30 h after transfection. Lysates were kept on ice for 30 min, followed by centrifugation for 30 min at 12,000 rpm at 4 °C. Then, the supernatants were mixed with 5× loading buffer (Mei5bio, MF145-01) before being separated by 6% SDS-PAGE gels (Beyotime, P0050A). Proteins were immunoblotted onto polyvinylidene difluoride membranes (Millipore), which were blocked in 5% nonfat milk dissolved in 150 mM NaCl, 10 mM Tris-Base (pH 7.4), and 0.1% Tween 20 at RT for 1 h. The membranes were then incubated with anti-HA (Cell signaling technology, H3724, 1:1000), anti-FLAG (Sigma–Aldrich, F7425, 1:1000), anti-TMEM63B (abcam, 1:1000), anti-β-COP (abcam, ab2899, 1:1000), anti-Gapdh (Bioworld, MB001, 1:10,000) primary antibodies, and horseradish peroxidase–conjugated secondary antibodies (Bioworld). The reactive protein bands were developed by ECL kit (Tanon, 180–501) and captured by a chemiluminescent imaging system (Tanon). To quantify protein expression, the integrated absorbances of protein bands were measured using ImageJ software (National Institutes of Health).
7
Western Blot Analysis of Protein Extracts
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The cells and tissues were lysed using RIPA lysis buffer containing 1% protease inhibitors (EpiZyme, Shanghai, China). Then the extracted supernatant was added to the 5 × loading buffer and incubated in a metal bath at 100 °C for 10 min. The protein concentration was detected using a protein quantitative detection kit (Beyotime, China). The protein samples were separated by 7.5% or 10% SDS-PAGE (EpiZyme, China), transferred to PVDF membranes (Millipore, Germany), and blocked by 5% skimmed milk powder for 2 h. Subsequently, the membranes were incubated with primary and secondary antibodies, respectively. The details of the antibodies referred are listed in Supplementary Table S2 . Finally, protein signals were visualized with an enhanced chemiluminescence kit and captured by A Tanon5200 Multiintelligent imaging system (Bio Tanon, China).
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