For immunoblot analysis, the cells were lysed in RIPA lysis buffer (50 mM Tris-HCL, 150 mM NaCl, 1%NP40, 0.5% Na deoxycholate, and 1 mM EDTA) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors (Beyotime). The total protein was extracted and followed by SDS-PAGE gel electrophoresis to analyze the expression of indicated proteins. For immunoprecipitation analysis, the cells were lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Roche) and phosphatase inhibitors (Beyotime). The cell lysates were incubated with primary antibodies overnight at 4 °C. Protein agarose A/G beads were then added for an additional 3 h. The beads were washed four times with RIPA lysis buffer and analyzed by immunoblot. The primary antibodies including anti-RAGA(D8B5) rabbit mAb (CST, 4357; dilution 1:1000), anti-CD47 rabbit mAb (Abcam, ab175388; dilution 1:1000), anti-CD47 mouse mAb (Abcam, ab3283; dilution 1:1000), anti-HA mouse mAb (Sigma, H9658; dilution 1:3000), anti-flag mouse mAb (Sigma, F1804; dilution 1:3000), anti-N-Cadherin rabbit mAb (CST, 13116; dilution 1:1000), anti-LAMP2[H4B4] mouse mAb (Abcam, ab25631; dilution 1:1000), anti-LAMP2A rabbit pAb (Abcam, ab18528; dilution 1:1000), anti-αTubulin mouse mAb (Abcam, ab7291; dilution 1:5000), anti-βactin mouse mAb (Sigma, A5316; dilution 1:5000) were used for immunoblot and immunoprecipitation assay.
Phosphatase inhibitor
Manufactured by Beyotime
Sourced in China, United States, Switzerland, Germany
Phosphatase inhibitor is a chemical compound used in laboratory settings to prevent the activity of phosphatase enzymes. Phosphatases are responsible for the removal of phosphate groups from various biomolecules, and their inhibition can be useful in maintaining the phosphorylation state of proteins and other targets during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Beyotime (47 mentions)
Pvdf membrane, by Merck Group (35 mentions)
Ripa lysis buffer, by Beyotime (29 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (26 mentions)
Ripa buffer, by Beyotime (25 mentions)
Bca protein assay kit, by Beyotime (23 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Bca kit, by Beyotime (7 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (6 mentions)
Gapdh, by Proteintech (5 mentions)
Protease inhibitor, by Roche (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (4 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Beyotime (4 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (4 mentions)
Lysis buffer, by Beyotime (4 mentions)
162 protocols using phosphatase inhibitor
1
Protein Expression and Interaction Analysis
2
Elucidating Ovarian Cancer Metabolic Pathways
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Ovarian cancer cell lines (A2780, A2780/DDP, COC1, COC1/DDP) were purchased from Zhejiang Chinese Medical University. The following reagents were used: phosphatase inhibitors (Biyuntian, S1873); CCK8 detection kit (Beyotime, C0037); DATS and cisplatin (Source leaf, B25320, and B24462); annexin VFITC kit (Nanjing KGI Bio, KGA108); glucose test kit (Nanjing built, F006–11); ROS activity detection kit (Nanjing built, E004–11); glutamine and glutamate determination kit (Sigma Aldrich, GLN1); and NAC, OM, AMPK inhibitor compound C, and AMPK activator AICAR (MCE, HYB0215, HYN6782, HY13418, and HY‐13417). The following antibodies were used: pAMPK (Bioss, bs4002R); AMPK, SIRT1, and PGC1α (Affinity, DF2656, DF6033, and AF5395); acetylated‐lysine (Cell Signaling Technology, 9441); and OXPHOS (Abcam, ab110411). All other reagents were purchased from Sigma Aldrich.
3
Protein Extraction from Liver Tissue
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Fresh liver tissue (100 mg) was taken and ground using liquid nitrogen. The total protein content was extracted from the liver tissues using a total protein extraction kit containing protease inhibitors (Biyuntian Biotechnology Co., Ltd., China) and phosphatase inhibitors (Biyuntian Biotechnology Co., Ltd., China) according to the manufacturer’s instructions. The protein concentration was determined by the BCA method (Biyuntian Biotechnology Co., Ltd., China). Total protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electropheresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk in Tris-buffered saline (1x) for 2 h, the membranes were probed with appropriate primary antibodies at 4°C overnight and then detected by HRP-labeled anti-rabbit or HRP-labeled anti-mouse secondary antibodies (Zhongshan Jinqiao Biotechnology Co, Ltd., China). The antigen-antibody complex was detected by an enhanced ECL reagent (Biyuntian Biotechnology Co., Ltd., China). After exposure, Image J was used for grayscale analysis.
4
Protein Extraction and Western Blot Analysis
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Cells were lysed with a solution containing lysis buffer (KeyGEN, China), protease inhibitors, phosphatase inhibitors, and PMSF (Beyotime, China) at 4°C for 30 min on a rocker. A BCA protein assay kit (KeyGEN, China) was used to quantify total protein. Protein samples were mixed with 5× buffer and boiled for 10 min before loading onto a 10% SDS-PAGE gel (Epizyme, China). After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, United States). Based on the position of the markers, the membranes were cut into pieces, blocked with QuickBlockTM blocking buffer (Beyotime, China) for 1 h, and incubated with primary antibodies (Supplementary Table S2 ) overnight at 4°C. The membranes were washed with TBST and then incubated with the corresponding secondary antibody (Supplementary Table S2 ) at room temperature for 1 h. The membranes were developed by ECL (Millipore, United States) using a ChemiDocTM Touch imaging system (Bio-Rad, United States). Intensity of the Western blot bands was quantified using Quantity One (United States).
5
Breast Cancer Cell Line Transfection and Protein Expression Analysis
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The breast cancer cell lines MDA-MB-231 and CAL-148 were transfected with the corresponding siRNA, CEP55, for 48 h. Cells were lysed with RIPA buffer containing a mixture of protease inhibitors and phosphatase inhibitors (Beyotime, Shanghai, China). After quantification and denaturation for each protein sample, 30 μg of protein was separated and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were exposed to a 5% skim milk solution for 2 h at room temperature, incubated with primary antibody (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C, then reacted with HRP-coupled secondary antibody for 2 h and developed using ECL substrate. Mouse monoclonal anti-GAPDH (Cell Signaling Technology, Beverly, MA, USA) was used as an internal standard.
6
Immunoprecipitation and Western Blot Analysis
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After transfection, the cells were cultured in a 100 mm dish for 48 h and then lysed using IP lysis buffer containing a mixture of protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology, P0013, Shanghai, China). Then, 1 µg of primary antibody was incubated with 30 µL of protein A+G beads on a rotator at room temperature for 2 h, the protein lysate was then added and incubated on the rotator at 4°C overnight. Next day, the beads and immune complexes were washed five times with IP lysis buffer by rotating at room temperature for five minutes at a speed (2000 rpm/min) of 20 s per round. The sample was added to the corresponding SDS loading buffer and heated at 95°C for 5 min. The immunoprecipitated samples were detected using western blotting.
7
Western Blot Analysis of Autophagy and Inflammasome Proteins
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Proteins were extracted using RIPA buffer containing protease inhibitors and phosphatase inhibitors (Beyotime, China). Samples were homogenized on ice, centrifuged for supernatant at 12,000 g for 15 min at 4°C and heated to 100°C for 5 min. Protein extracts resuspended in sample loading buffer were separated by electrophoresis through 12% polyacrylamide gels and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk (Sangon Biotech Shanghai Co.,Ltd., China), membranes were incubated with primary antibodies anti-LC3 (4108S, CST, USA; 1: 1,000 dilution), anti-Beclin 1 (3738, CST, USA; 1: 1,000 dilution), anti-NLRP3 (15101S, CST, USA; 1: 1,000 dilution), anti-NLRC4 (ab201792, abcam, UK; 1: 1,000 dilution), anti-GAPDH (BA2913, Boster, China; 1: 1,000 dilution), anti-Tubulin (AF1216, Beyotime, China; 1: 1,000 dilution) and anti-Histone H3 (ab194681, abcam, UK; 1: 1,000 dilution) overnight at 4°C. Membranes were then washed and incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (A0208, Beyotime, China; 1: 3,000 dilution) for 1 h at room temperature. Proteins were visualized using ECL luminescence reagent (Meilunbio, China). The gray-scale values of the bands were determined by Image J launcher broken symmetry software program (National Institutes of Health, Bethesda, MD, USA).
8
Quantification of mPFC Protein Levels
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mPFC tissues were kept in a buffer solution containing phosphatase inhibitors and protease inhibitors (Beyotime, Shanghai, China) to prepare a cell suspension. The protein solution was collected by centrifuge (1200×g, 10 min). The protein concentration was quantified by the BCA kit (Beyotime, Shanghai, China) following the manual instruction. Before blotting assays, the protein solution was diluted and boiled at 100 °C for 10 min. Protein samples were separated by SDS–PAGE under 80 V and 120 V electric fields. After transferring the PVDF membrane, 5% fetal bovine serum (BSA) was applied for 2 h blocking, followed by primary antibody incubation at 4 °C overnight. The membrane was subsequently washed with 0.01% TBST and incubated with a secondary antibody for 2 h. Protein bands were visualized using an imaging system (Bio-Rad, Hercules, USA), Integrated gray values of each band were measured using ImageJ (National Institutes of Health, Bethesda, USA).
9
Immunoprecipitation and Western Blot
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Cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 0.5% NP-40) containing 1 mM PMSF, 1 mM phosphatase inhibitors (Beyotime Biotechnology), and protease inhibitors (Roche) and incubated on ice for 30 minutes. Cell lysates were centrifuged for 10 minutes (120,000g, 4°C). Equal amounts (50 μg) of proteins from each sample were boiled in loading buffer as input protein. The rest of lysates were incubated with the primary antibody for 6–8 hours at 4°C. Protein A/G magnetic bead was added to the incubation mixture for an additional 2–3 hours to pull down antibody-protein complexes. The immunoprecipitates were collected and washed 3 times with PBST buffer before being resolved by SDS-PAGE.
10
Assessment of GSK-3β and A1R in Cerebral Ischemia
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24 hours after reperfusion, the animals were euthanized with excessive chloral hydrate, and the hippocampus was rapidly separated on infarcted side. The operation should be performed on a cold surface, and liquid nitrogen was used to terminate the cell metabolism. The tissues were homogenized in 100 RIPA lysis buffer (Beyotime, CHN): 10 phosphatase inhibitors (Roche, Germany): 1 PMSF (Beyotime, CHN) on ice. Tissue extract was centrifuged at 12000g at 4°C for 30 min. Western blotting was performed with standard procedures. PVDF membranes were incubated with GSK-3β polyclonal antibody, phospho-GSK-3β (Ser-9) polyclonal antibody (1 : 1000 dilution, Affinity Biosciences, USA), adenosine A1 receptor polyclonal antibody (1 : 1000 dilution, Abcam, USA), and GAPDH polyclonal antibody (1 : 1000 dilution, Hangzhou Xianzhi, CHN) at 4°C for 16 hours. After that, the membranes were incubated for 1 hour with the horseradish peroxidase- (HRP-) conjugated goat anti-rabbit (1 : 5000 dilution, Biosharp, CHN) at room temperature.
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