Cells were lysed using RIPA buffer (Biosesang Inc, Seongnam, Korea) containing phosphatase and protease inhibitor cocktail (GeneDEPOT, Barker, TX, USA). Protein concentration was measured using Pierce™ 660 nm protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane (Merck, Kenilworth, NJ, USA). After blocking with 5% skim milk for 1 h, membranes were incubated overnight at 4 ℃ with primary antibodies, which were dissolved in PBST containing 5% BSA. The following primary antibodies were used: anti-Mindin (SPON2) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)], anti-RBP-Jk (Santa Cruz Biotechnology), and anti-activated Notch1 (Abcam, Cambridge, UK), which detected Notch1 intracellular domain (N1ICD). Anti-GAPDH (Bioworld, Dublin, OH, USA) was used as control. Further, membranes were incubated with HRP-conjugated secondary antibody (Bethyl Laboratories, Montgomery, TX, USA) for 90 min followed by detection using ECL kit (Bio-Rad, Hercules, CA, USA) and Supernova-Q1800.
Ripa buffer
Manufactured by Biosesang
Sourced in United States, Cameroon, Germany
RIPA buffer is a detergent-based buffer solution commonly used in biological research for cell lysis and protein extraction. It contains a combination of ionic and non-ionic detergents, as well as other components that aid in the solubilization and extraction of proteins from cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (11 mentions)
Anti β actin, by Santa Cruz Biotechnology (9 mentions)
β actin, by Cell Signaling Technology (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Gapdh, by Cell Signaling Technology (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Skim milk, by BD (6 mentions)
P akt, by Cell Signaling Technology (5 mentions)
P erk, by Cell Signaling Technology (5 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (3 mentions)
Ponceau s staining solution, by Avantor (3 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
β catenin, by Cell Signaling Technology (3 mentions)
Amersham imager 600, by GE Healthcare (3 mentions)
81 protocols using ripa buffer
1
Western Blot Analysis of Notch Pathway
2
Protein Expression Analysis in Pancreatic Cells
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The pancreas and pancreatic acinar cells were lysed with a radioimmunoprecipitation assay (RIPA) buffer (Biosesang, Gyeonggi‐do, Korea) containing protease and phosphatase inhibitor cocktails (GenDepot, Barker, TX). The proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto nitrocellulose membranes. The blots were immunostained with the appropriate primary antibodies followed by secondary antibodies conjugated to horseradish peroxidase. Antibody binding was detected with an enhanced chemiluminescence reagent (Bio‐Rad. Hercules, CA, USA). The primary antibodies against the following factors were used: ANGPTL4 (Thermo Fisher Scientific, Waltham, MA, USA, cat# 40‐9800), GAPDH, and β‐actin (Santa Cruz Biotechnology, Dallas, TX, USA, cat# sc‐47724, and sc‐47778). The secondary antibodies were purchased from Santa Cruz Biotechnology.
3
Total RNA and Protein Extraction from Gastric Cancer Cells
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Total RNA from gastric cancer cells (2×106 cells/well) in 100 mm cell culture dish was prepared using Trizol reagent according to the manufacturer’s protocols (Invitrogen). Protein sampling was collected in RIPA buffer (Biosesang) containing a protease inhibitor cocktail (Sigma) on ice for 30 min and were passed through an 18-gauge needle, and spin down. The supernatant was analyzed for protein content using the BCA method (Thermo Scientific).
4
Decellularized Extracellular Matrix Proteome Analysis
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The RdECM was lysed using RIPA buffer (Biosesang, Seongnam, Korea) supplemented with a proteinase inhibitor cocktail (Sigma), and the protein content was measured using a BCA protein assay kit (Pierce, Waltham, MA, USA). Then, the samples were treated with 8 M urea and 50 mM ammonium bicarbonate buffer for 3 h, 1 M dithiothreitol treatment for 2 h, 550 mM iodoacetamide for 1 h, and an additional 1 M dithiothreitol treatment for 30 min. Next, the samples were incubated with trypsin at 37 °C for 18 h, followed by desalting with Sep-Pak. Mass spectrometric analysis was performed using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap instrument (Thermo Fisher Scientific, Waltham, MA, USA). According to the manufacturer’s instruction. The mass data were acquired automatically using Proteome Discoverer 2.4 (Thermo Scientific, USA) against the porcine protein UniProt database. The acquired data were categorized based on the PANTHER gene ontology (GO) classification system. The collected data were also annotated with a human Matrisome database (MatrisomeDB, http://matrisomeproject.mit.edu/ ).
5
Western Blot Analysis of Treated Cells
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Treated cells were washed with ice-cold PBS (Welgene, Gyeongsan, Korea), scraped in 150 μL of RIPA buffer (Biosesang, Seongnam, Korea), and further incubated on ice for 30 min. Cell debris was removed by centrifugation at 15,000 rpm for 15 min. Equal amounts of total protein determined by BCA protein assay (Thermo Scientific, Waltham, MA, USA) were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Each membrane was blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 and incubated with primary antibodies overnight at 4 °C. Blots were developed with an HRP-conjugated secondary antibody, followed by visualization using enhanced chemiluminescence (ATTO KOREA, Daejeon, Korea).
6
Molecular Mechanisms of STAT1 Activation
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The THP-1 cells were harvested and lysed after treatment with compounds. The proteins were extracted using an RIPA buffer (BIOSESANG, Seongnam, Korea) containing protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA). Protein lysates were electrophoresed on 9% SDS-PAGE gels and transferred onto PVDF membranes. The membrane was blocked with 5% BSA in Tris-buffered saline with Tween-20 (TBST; BIOSESANG) solution for 1 h at room temperature, followed by washing with TBST. Membranes were incubated with the following antibodies: (1) at 1:1000 dilution—phosphorylated STAT1(Tyr701) (#7649; Cell Signaling, Danvers, MA, USA), phosphorylated TBK1 (Ser172) (#5483; Cell Signaling), phosphorylated IRF3 (Ser396) (#4947S; Cell Signaling), phosphorylated STING (S366) (#19781; Cell Signaling), STAT1 (#9172; Cell Signaling), IRF3 (#11904; Cell Signaling), TBK1 (#3504; Cell Signaling), STING (#13647; Cell Signaling), and (2) at 1:3000 dilution—β-actin (#4970; Cell Signaling). HRP-conjugated secondary antibody (#7074; Cell Signaling) was used at a dilution of 1:3000. Luminescent images were visualized using the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).
7
Western Blot Analysis of DW2282 and KAS-08
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THP-1 cells were incubated with the indicated concentrations of DW2282 or KAS-08 for 3 h. After harvesting, the cells were divided into equal volumes and heated at a specific temperature for 3 min. Proteins were extracted with an RIPA buffer (BIOSESANG) containing protease and phosphatase inhibitor cocktail (Thermo Fisher). Protein lysates were electrophoresed on 9% SDS-PAGE gels and transferred onto PVDF membranes. The membrane was blocked with 5% BSA in Tris-buffered saline with Tween-20 (TBST) solution for 1 h at room temperature, followed by washing with TBST. The membranes were analyzed according to the above-mentioned Western blot analysis protocol.
8
Western Blot Analysis of Signaling Proteins
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MIAPaCa-2 and PANC-1 cells were washed with Dulbecco’s phosphate buffered saline (DPBS) and lysed with RIPA buffer (Biosesang, Korea) containing 1% Triton X-100, Xpert protease inhibitor, and phosphatase inhibitor Cocktail (GenDEPOT, TX, USA). Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, MA, USA). Protein transfer was verified using the Ponceau S staining solution (Amresco, OH, USA), and the blots were then incubated with the appropriate primary (1:500, except for β-actin [1:10,000]) and the secondary antibodies (1:1,000, except for β-actin [1:20,000]) conjugated to HRP. Antibody binding was detected using an enhanced chemiluminescence reagent (Bio-Rad, CA, USA) using primary antibodies specific to the proteins of interest, and the proteins were detected using X-ray film and enhanced chemiluminescence reagent. Primary antibodies were used against the following: p-ERK, ERK, p-AKT, AKT, and β-actin (Cell Signaling Technologies, MA, USA) and p-BRAF (Santa Cruz Biotechnology, TX, USA), and the secondary antibodies were purchased from Cell Signaling Technologies.
9
Protein Extraction and Western Blot Analysis
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For protein extraction, the tissues were sonicated on ice in RIPA buffer (Biosesang, Seongnam, Korea) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). After centrifugation at 12,000 rpm at 4 °C for 20 min, cell lysates were prepared and analyzed for protein concentration by Bradford assay. A total of 40 μg protein was fractionated by SDS-PAGE on 8–16% Tris–glycine gel (Komabiotech, Seoul, Korea), transferred to PVDF membrane (Thermo Fisher Scientific) and blotted with antibodies against H2 antigen (1:500, Cat No. 78438, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or β actin (1:1000, Santa Cruz Biotechnology) [14 (link)].
10
Protein Extraction and Western Blot Analysis
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To extract the protein, cells were lysed in the radioimmunoprecipitation assay (RIPA) buffer (Biosesang, Seongnam-si, Republic of Korea) for 30 min on ice and the cell lysate was centrifuged at 15,000 × g for 20 min at 4°C. The concentration of total protein in the collected supernatant was measured using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (20 μg) were loaded onto SDS-PAGE (10–12%) gels and electrophoresed for separation followed by transfer onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked in 5% skim milk (BD Bioscience, Franklin Lakes, NJ, USA) in TBS-T (0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA)) buffer for 30 min and incubated with primary antibody at 4°C overnight. The membranes were then washed with TBS-T buffer and incubated with HRP-linked secondary antibody for 1 h at room temperature. Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Burlington, MA, USA) was used for chemiluminescence detection.
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