Protein Extraction from gastric tissue samples using RIPA buffer (50mg/400μl, Cat.C500008–0010, Sangon Biotech, Shanghai) and homogenized with an automated tissue tearor (Precellys Evolution Homogenizer, Thermo Scientific Fisher). Cells lysis containing RIPA lysates were removed from the 6-well plate culture dish using cell scraper on ice. The protein concentration was detected by the Bradford method. 20ug equivalent protein was loaded, the gel was run at 100V for 60 minutes and transferred to a nitrocellulose membrane (PVDF membrane with pore size 0.2um/0.45μm, Thermo Fisher Scientific) through SDS-PAGE electrophoresis and immunoblotting with the Pin1 and its substrate antibodies.
Ripa buffer
Manufactured by Sangon
Sourced in China
RIPA buffer is a common lysis buffer used in protein extraction and western blotting applications. It contains a combination of ionic and non-ionic detergents that help solubilize cellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Bca protein assay kit, by Sangon (4 mentions)
Protease inhibitor, by Sangon (4 mentions)
Pierce phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Proteinase inhibitor, by Roche (2 mentions)
Pvdf membrane, by Roche (2 mentions)
Bca protein assay kit, by Abcam (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Gapdh, by Proteintech (2 mentions)
Ab8245, by Abcam (2 mentions)
Mbs435036, by MyBioSource (2 mentions)
Ab9485, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
53 protocols using ripa buffer
1
Pin1 Protein Extraction from Gastric Tissue
2
Western Blot Analysis of EMT Markers
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Cells were lysed using the RIPA buffer (Sangon, China) containing a Proteinase inhibitor (Roche, Switzerland) and a Pierce phosphatase inhibitor (Thermo Fisher, USA) 27 (link). Protein extracts were boiled in a loading buffer followed by separation on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis platform (SDS-PAGE). Separated protein bands were transferred onto polyvinylidene fluoride membranes. Primary antibodies against EPAS1 (7096s, CST, USA), GAPDH (2118S, CST, USA), N-cadherin (13116S, CST, USA), Vimentin (5741S, CST, USA), E-cadherin (3195S, CST, USA), MMP-9 (13667S, CST, USA), MMP-2 (40994S, CST, USA), Snail (3879S, CST, USA), and Twist (69366S, CST, USA) were diluted at a ratio of 1:1000 in accordance with the manufacturer's instructions. Then, membranes were probed with a goat anti-rabbit IgG highly cross‐adsorbed secondary antibody (HSA0003, maibio, China) for 2 h at room temperature after which they were exposed to the enhanced chemiluminescence (ECL) reagent for visualization.
3
Western Blot Analysis of ROCK1 Protein
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RIPA buffer (Sangon Biotech Co., Ltd) was used to extract total proteins from U2OS cells. Protein concentrations were measured using a bicinchoninic acid assay kit. Protein samples were boiled for 5 min for denaturation. Subsequently, 10% SDS-PAGE was used to separate the proteins with 30 µg protein per lane. The proteins were transferred to PVDF membranes, followed by blocking for 2 h in PBS containing 5% FBS (Sigma-Aldrich; Merck KGaA) at room temperature. The membranes were probed with rabbit anti-ROCK1 (1:800; cat. no. ab97592; Abcam) and anti-GAPDH (1:800; cat. no. ab37168; Abcam) for 12 h at 4°C, followed by incubation with horseradish peroxidase-conjugated goat secondary antibody (IgG; 1:1,000; cat. no. ab6721; Abcam) for 2 h at 24°C. Signal development was performed using the ECL Western Blotting Substrate kit (cat. no. ab65623; Abcam), and the data were processed using Image J v.1.48 software (National Institute of Health).
4
Histone H3 citrullination analysis
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Pancreatic samples were snap-frozen and homogenized in RIPA buffer (Sangon Biotech, Shanghai, China) on ice. After centrifugation at 12000 rpm for 10 min at 4°C, the protein content of the supernatant was mixed with loading buffer. An equal amount of protein per sample was resolved on gradient gels (15% Tris-glycine gels) and electroblotted on PVDF membranes, which were then incubated with primary antibodies (rabbit polyclonal anti-H3cit, Abcam; rabbit polyclonal anti-GAPDH, Servicebo) at 4°C overnight and subsequently with appropriate HRP-conjugated secondary antibodies (Servicebo, Wuhan, China) for 1 h at room temperature. The blots were developed with enhanced chemiluminescence substrate (Sangon Biotech, Shanghai, China). Blots were quantified using ImageJ software.
5
Western Blot Protocol for TFAP2B Detection
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Cells were lysed in ice-cold RIPA buffer (Sangon Biotech Co., Ltd.) and the protein was quantified using a BCA Protein Assay kit (Abcam). Lysates (20 µg/sample) were loaded on 8–12% denaturing SDS-PAGE gels and transferred to a PVDF membrane (Roche Diagnostics GmbH). Tris-HCl buffer containing 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) was used to block the membranes at 28°C for 2 h. The membrane was probed with the primary antibodies prepared in Tris-HCl buffer containing 5% BSA at 4°C overnight. Subsequently, the membranes were washed three times with Tris-HCl buffer containing 0.1% Tween-20, followed by incubation with the appropriate secondary antibodies prepared in Tris-HCl buffer at 28°C for 1 h. Finally, the membranes were washed three times, detected and visualized by an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.). The antibodies used in this study included anti-TFAP2B (1:500; cat. no. 13183-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:5,000; cat. no. YM3029; ImmunoWay Biotechnology Company), HRP-conjugated goat anti-rabbit IgG (1:1,000; cat. no. 7074; Cell Signaling Technology, Inc.) and HRP-conjugated rabbit anti-mouse IgG (1:1,000; cat. no. 7076; Cell Signaling Technology, Inc.).
6
Quantification of Caspase-1 Activation in P. aeruginosa-infected PMs
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PMs were infected with
P.
aeruginosa for 6 h, and the supernatants were treated with methanol and chloroform with vortexing and centrifugation at 10,000
g for 5 min to obtain total proteins. Cell lysates were obtained by lysing PMs in RIPA buffer (Sangon Biotech, Shanghai, China). Equal amounts of total proteins extracted from supernatants and lysates of PMs were diluted in 5× SDS-PAGE loading buffer and boiled for 5 min. Proteins were separated on 12% polyacrylamide gels. Following electrophoretic transfer of protein onto PVDF membranes (Millipore, Billerica, USA), membranes were blocked in 5% skim milk. The membranes were incubated with mouse anti-caspase-1 monoclonal antibody (Adipogen, San Diego, USA) at 4ºC overnight. Then, the membranes were incubated with HRP-conjugated mouse antibody (Beyotime, Shanghai, China) for 1 h at room temperature, followed by visualization using a chemiluminescent substrate (Thermo Scientific).
P.
aeruginosa for 6 h, and the supernatants were treated with methanol and chloroform with vortexing and centrifugation at 10,000
g for 5 min to obtain total proteins. Cell lysates were obtained by lysing PMs in RIPA buffer (Sangon Biotech, Shanghai, China). Equal amounts of total proteins extracted from supernatants and lysates of PMs were diluted in 5× SDS-PAGE loading buffer and boiled for 5 min. Proteins were separated on 12% polyacrylamide gels. Following electrophoretic transfer of protein onto PVDF membranes (Millipore, Billerica, USA), membranes were blocked in 5% skim milk. The membranes were incubated with mouse anti-caspase-1 monoclonal antibody (Adipogen, San Diego, USA) at 4ºC overnight. Then, the membranes were incubated with HRP-conjugated mouse antibody (Beyotime, Shanghai, China) for 1 h at room temperature, followed by visualization using a chemiluminescent substrate (Thermo Scientific).
7
Western Blot Analysis of Cell Signaling Pathways
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Protein was extracted using RIPA buffer (Sangon Biotech, Shanghai, People’s Republic of China) containing proteinase inhibitors (MedChem Express) and phosphatase inhibitors (MedChem Express). The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). The following antibodies were used for Western blot: anti-CDK7 (Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (Cell Signaling Technology), anti-poly(ADP-ribose) polymerase (anti-PARP) (Cell Signaling Technology), anti-cleaved PARP (Cell Signaling Technology), anti-RNA Pol II (Abcam, Cambridge, UK), anti-phospho RNA Pol II (Ser2) (Abcam), anti-phospho RNA Pol II (Ser5) (Abcam), anti-phospho RNA Pol II (Ser7) (Millipore), anti-CDK1 (Abcam), and anti-CDK1 (phospho T161) (Abcam).
8
Western Blot Analysis of Colon Proteins
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Colon proteins were extracted as described previously (25 (link)). Briefly, RIPA buffer supplemented with protease inhibitors (Sangon Biotech Co., Ltd.) was used as lysis buffer. Colon proteins were separated by 10% SDS-PAGE, and the gels were then electro-transferred onto nitrocellulose filter membranes (Whatman; Cytiva). The membranes were incubated with antibodies against α7nAChR (Sigma-Aldrich; Merck KGaA, cat. no. M220, 1:1,000), phosphorylated (p-)SHP2 (Abcam; cat. no. ab62322, 1:500), SHP2 (Abcam, cat. no. ab131541, 1:500), p-STAT3 (Cell Signaling Technology, Inc.; cat. no. 9145, 1:1,000); STAT3 (Santa Cruz Biotechnology, Inc., cat. no. sc-482, 1:100), p-Jak2 (Abcam, cat. no. ab32101, 1:1,000); Jak2 (cat. no. ab108596, Abcam, 1:1,000) or GAPDH (Abcam, cat. no. ab181602, 1:2,000) overnight at 4°C. The membranes were then incubated with an IRDye 800CW-conjugated secondary antibody (Rockland Immunochemicals Inc. 1:20,000) for 1 h at room temperature. Images were acquired using an Odyssey infrared imaging system (Odyssey® CLx Imaging System, LI-COR Biosciences) and ImageJ software v1.52 (NIH) was used for analysis.
9
Western Blot Analysis of CILP Protein
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The NP tissues and the differently treated NP cells were lysed with RIPA buffer (Sangon Biotech, Shanghai, China). Next, the proteins were quantified by a BCA Protein Assay Kit (Abcam, Cambridge, UK), and then the proteins with different molecular weights were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred into polyvinylidene fluoride (Millipore, Massachusetts, USA) membranes. The above membranes were incubated with 5% skim milk for approximately 1 h and further incubated with specific primary antibodies, including anti-CILP (Abcam, 1: 1000 dilution, ab192881) and anti-β-actin (Abcam, 1: 1000 dilution, ab8227) at 4 °C overnight. After being washed about two times, the membranes were further incubated with the secondary antibody (Abcam, 1: 2000 dilution, ab205718) for about 1 h at room temperature. Ultimately, the enhanced chemiluminescence reagents (Millipore, Boston, Massachusetts, USA) and Image J (National Institutes of Health, Bethesda, USA) were applied to observe and analyze all the protein bands.
10
Western Blot Analysis of Notch Pathway
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RIPA buffer (Sangon Biotech) was used to extract total protein. After separated using SDS-PAGE, proteins were transferred onto PVDF membranes, then blocked 1 h. The membranes were incubated with primary antibodies (Cell Signaling Technology, Danvers, MA, USA): Notch1, JAG1, and HES-1 or GAPDH at 4°C for a whole night. After HRP-conjugated anti-rabbit secondary antibody was used to incubate the membranes for 2 h, the signal was evaluated using the ECL system.
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