Specimen from aortic valves were cut into pieces and smashed with liquid nitrogen. Protein extracted from aortic valve was then lysed in RIPA Lysis (Beyotime, Jiangsu, China) and Extraction Buffer (Beyotime) containing phenylmethanesulfonyl fluoride (PMSF, Beyotime) for 30 mins on ice. After 12,000 g centrifugation for 10 mins at 4 °C, the supernatants were then transferred to new Eppendorf tubes. The concentration of protein was measured using the BCA method and whole cell lysates were then subjected to western blot using standard procedures.
Extraction buffer
Manufactured by Beyotime
Sourced in China
Extraction buffer is a solution used to extract and isolate target molecules, such as proteins, DNA, or RNA, from biological samples. It is designed to disrupt cellular structures, denature unwanted molecules, and maintain the stability and integrity of the target analyte. The exact composition and formulation of the extraction buffer may vary depending on the specific application and the nature of the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis, by Beyotime (2 mentions)
β actin, by Proteintech (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Immobilon hrp substrate, by Merck Group (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Anti gapdh mouse monoclonal antibody, by Proteintech (1 mentions)
Membrane and cytosol protein extraction kit, by Beyotime (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
β catenin, by Proteintech (1 mentions)
C myc, by Proteintech (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Colorimetric assay kit, by Abcam (1 mentions)
Odyssey system, by LI COR (1 mentions)
Ab252897, by Abcam (1 mentions)
Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg antibody, by Santa Cruz Biotechnology (1 mentions)
13 protocols using extraction buffer
1
Aortic Valve Protein Extraction and Analysis
2
Western Blot Protein Extraction and Analysis
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The collected cells were efficiently lysed by RIPA Lysis (Beyotime, Shanghai, China) and the proteins were extracted with Extraction Buffer (Beyotime, Shanghai, China) in accordance with the manufacturer’s instructions. The protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), was added to block the endogenous proteolysis. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. Nonspecific reaction was blocked with 5% skimmed milk in Tris-buffered saline Tween (TBST) buffer. Anti-PABPC1 rabbit monoclonal antibody (Abcam, Cambridge, UK) and anti-GAPDH mouse monoclonal antibody (Proteintech, Rosemont, IL, USA) were used as primary antibodies, respectively, at a ratio of 1:2000. After washing three times with TBST buffer, the membranes were incubated with HRP-conjugated secondary antibody (Proteintech, Rosemont, IL, USA) at a ratio of 1:5000. Protein expression was detected with the commercial ECL kit (Thermo, Waltham, MA, USA), and analyzed with IMAGE J software (v1.8.0, Bethesda, MD, USA).
3
Cell Lysis and Membrane Protein Extraction
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For total cell lysis, cells were lysed in extraction buffer (Beyotime, Hangzhou, China) for 1 h on ice. The lysates were centrifuged at 12,000×g for 20 min. For extracting the membrane protein, a membrane and cytosol protein extraction kit (Beyotime, Hangzhou, China) was used following the instructions of the manufacturer. The protein concentration was quantified by bicinchonininc acid (BCA) assay. Western blots were performed as previously described61 (link).
4
Quantification of Epithelial Markers
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Total protein of the cells was extracted by extraction buffer (Beyotime, China) and then the BCA Protein Assay Kit (Pierce Biotechnology) was used to assess the protein concentration. Then lysates were loaded in 10% SDS-PAGE gel and separated after electrophoresis. Then the protein was transferred from the gel to a NC membrane. The membrane then get blocked in 5%BSA for an hour. Primary antibodies WNT7A (ab217844, abcam, UK), c-Myc, β-catenin, N-cadherin, E-cadherin and β-actin (Proteintech, US) were used to probe the specific target protein overnight at 4 °C and then bound with the species-specific secondary antibodies (Proteintech, US) for an hour at room temperature. The concentration of target proteins were determined by Odyssey imaging system (LI-COR Biosciences, Lincoln, NE). All the western blot experiments are repeated 2–3 times.
5
Caspase-3 Activity Assay in Rat SN
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The activity of caspase-3 in the SN of rat was measured by a colorimetric assay as described [17 ]. In brief, the SN of rats were lysed in extraction buffer (Beyotime Inc.), and the activity of caspase-3 was analyzed with a colorimetric assay kit (Abcam Inc.) following the manufacturer's instructions.
6
Cytosolic Cytochrome c and Caspase 3 Analysis
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SMG-C6 cells were treated as described above in Section 2.4 . Cells were lysed, and total or cytosolic protein was extracted using the corresponding extraction buffer (Beyotime, Shanghai, China). Western blot analysis of cytosolic cytochrome c (host: rabbit; 1:2000 dilution, Proteintech, Wuhan, China), cleaved caspase 3 (host: rabbit; 1:1000 dilution, CST, MA, USA), and β-actin (host: mouse; 1:20,000 dilution, Proteintech, Wuhan, China) was performed following a standard procedure. Secondary antibodies were DyLight 680-labeled goat anti-rabbit and DyLight 800-labeled goat anti-mouse (Thermo Scientific, MA, USA). The bands were detected with an Odyssey system (LI-COR, Lincoln, NE, USA), and the image analysis was performed using ImageJ (Fiji, Version 1.0). β-actin was utilized as an internal standard.
7
Caspase-3 Activity Quantification
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After the indicated treatment for 24 hours, neurons were lysed in extraction buffer (Beyotime Biotechnology, Shanghai, China), as previously described (Gao et al., 2016). The caspase-3 activity was quantified using a fluorometric assay kit (ab252897, Abcam), in accordance with the manufacturer's instructions.
8
Western Blot Analysis of EZH2 Expression
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Total protein was extracted from AMC-HN-8 cells and EZH2-overexpressing cells with radioimmunoprecipitation assay lysis and extraction buffer (Beyotime Institute of Biotechnology, Haimen, China). Western blotting was performed as previously described (10 (link)) using an anti-KMT6/EZH2 antibody (rabbit polyclonal; dilution, 1:1,000; catalog no., ab3748; Abcam) and a mouse anti-human β-actin antibody (dilution, 1:1,000; catalog no., A5441; Sigma-Aldrich, St. Louis, MO, USA). The secondary antibody was a goat anti-rabbit IgG horseradish peroxidase-conjugated antibody (dilution, 1:2,000; cat no. sc-2030; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membrane was developed using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Inc.).
9
Bladder Tissue Protein Isolation and Analysis
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Total protein was isolated from the bladder tissues. Briefly, freshly isolated bladder tissue was lysed in ice-cold extraction buffer (Beyotime Biotechnology, Nantong, China). Protein concentration was determined by the Bradford method. Protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Pall, Ann Arbor) by standard procedures. Transferred blots were incubated sequentially with antibodies, including primary rabbit anti-TM (1 : 200), primary mouse anti-β-actin (1 : 3000), and HRP-conjugated secondary antibodies (1 : 3000). Protein bands were visualized with an enhanced chemiluminescence detection kit and recorded on radiographic film (FluorChem FC2, Alpha Innotech). The resulting images were analyzed with ChemiImager 4000 to determine the integrated density value of each protein band.
10
Quantification of Hepatic and Cellular Triglycerides
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Liver tissue was broken down using Micro Tissue Grinders (Tiangen Biotech, Beijing, China) with an extraction buffer (Beyotime Biotech, Haimen, China). The tissue suspension was centrifuged at 3000× g for 10 min to obtain supernatant, which was used to detect the TG content using a tissue triglyceride assay kit (JianCheng), following the manufacturer’s instructions. Bovine hepatocytes were collected from the 6-well plates after administration. The cells were lysed using a lysis buffer (Sangon Biotech, Shanghai, China) in an ice bath. The lysate was centrifuged at 12,000× g at 4 °C for 5 min, and the supernatant was collected for triglyceride analysis using the tissue triglyceride assay kit (JianCheng), according to the manufacturer’s protocol. Total protein concentration was estimated by the BCA method (Applygen, Beijing, China) and performed according to the manufacturer’s instructions. Hepatic and cellular TG were normalized to total protein contents.
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