The LX-2 cells were seeded into a 6-well plate and were treated with different concentrations of CD147 0.125, 0.25, 0.5 and 1.0 μg/ml). Following 24 h treatment, the medium was removed and the cells were harvested using radioimmunoprecipitation (RIPA) cell lysis buffer (Beyotime Institute of Biotechnology, Nantong, China). For the tissue specimens, the liver tissues were collected from rat models of hepatic fibrosis induced by carbon tetrachloride (CCl4; The Third Chemical Reagent Factory, Tianjin, China). The tissues were cut into small sections and the cells were disrupted using a tissue homogenate method. Briefly, the tissue sections were added to RIPA, placed on ice, and subsequently homogenized using a pro200 Homogenizer (Pro Scientific, Inc., Oxford, CT, USA). The total protein was extracted from 50 mg tissue samples and LX-2 cells in a 6-well plate using RIPA cell lysis buffer, and the protein concentration was measured using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Western blotting was performed, according to a standard method.
Ripa cell lysis buffer
Manufactured by Beyotime
Sourced in China
RIPA cell lysis buffer is a chemical solution used for extracting proteins from cells. It contains a combination of detergents, salts, and buffering agents that help to disrupt cell membranes and solubilize cellular proteins. The buffer is designed to maintain the native conformation and activity of the extracted proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (16 mentions)
Pvdf membrane, by Merck Group (14 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Chemiluminescent hrp substrate, by Merck Group (3 mentions)
Bca kit, by Beyotime (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Phosphatase inhibitor, by Merck Group (3 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (3 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Nitrocellulose membrane, by Pall Corporation (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Westernbright ecl hrp substrate, by Advansta (2 mentions)
Sds page, by Solarbio (2 mentions)
Bicinchoninic acid quantification kit, by Beyotime (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
85 protocols using ripa cell lysis buffer
1
Evaluating CD147 Effects on Hepatic Fibrosis
2
Protein Extraction and Western Blot Analysis
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The cells were washed with chilled PBS and re-suspended in RIPA cell lysis buffer (Beyotime) completely for 30 min incubation on ice. And the tumor tissue samples were homogenated in RIPA cell lysis buffer completely. Then, they were centrifugated at 12, 000 g for 15 min at 4°C. The supernatant was then stored at -80°C for following research. The concentrations of protein were measured using bicinchonininc acid (BCA) kit (Thermo scientific). 30 μg/lane proteins were then separated on SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride (PVDF, Bio-Rad, CA, USA) membrane blocked with 5% slim milk in TBS buffer supplemented with 0.1% Tween-20 (Beyotime). Then the primary antibodies were used for incubation overnight at 4°C. Then secondary antibodies, including goat anti-mouse and goat anti-rabbit (Cell Signaling Technology, MA, USA), were used to horseradish peroxidase for 2 h at room temperature. The bound antibodies were observed through incubating membranes using chmilumiescence reagents (Thermo scientific) ahead of X-ray film (Eastman Kodak Company, NY, USA). The protein levels were quantified with Image J software (National Institutes of Health, Bethesda, MD). The primary antibodies used in our study were showed in Table 1 .
3
Protein Quantification and Western Blot Analysis
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The concentration of protein was measured by a bicinchoninic acid protein assay kit (Invitrogen, Shanghai, China) after cells and tissue samples were processed in RIPA cell lysis buffer (Beyotime, Shanghai, China). The proteins were loaded onto a 10% SDS-PAGE for electrophoresis and then transferred to 0.22 m PVDF membranes (Millipore, MA, USA). Membranes were blocked with blocking buffer (1 × TBST with 5% skim milk), incubated with the appropriate primary antibodies, and treated with the secondary antibody. Immobilon Western Chemiluminescence HRP Substrate (Solarbio, Beijing, China) was then used to visualiza the brands.
4
Extracellular Vesicle Isolation and Analysis
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After 48 h of transfection with OPTI-MEM (no FBS), the culture medium was harvested and centrifuged at 5,000 × g for 5 min at 4°C to remove dead cells, followed by 16,000 × g for 10 min at 4°C for further removal of cellular debris. The collected culture medium was concentrated using Amicon Ultra15 Centrifugal Filters (10K, Millipore, MA, United States, Cat# UFC801008) by centrifugation at 4,500 × g for 30 min at 4°C. Adhered cells were washed twice with ice-cold PBS, followed by resuspension in RIPA cell lysis buffer (Beyotime, Cat# P0013B) containing the proteinase inhibitor PMSF (1:100, Beyotime, Cat# ST505) and the phosphatase inhibitor PhosSTOP (Roche, Mannheim, Germany, REF 04906845001). After incubation on ice for 30 min and centrifugation, the supernatants (soluble cell fraction) were carefully separated from the pellets (insoluble cell fraction). For denaturing SDS‒PAGE, aliquots of culture medium and cell fractions were treated with 4× protein loading buffer containing β-mercaptoethanol and SDS and boiled for 10 min. For nondenaturing PAGE, samples were treated with 4× protein loading buffer without β-mercaptoethanol, SDS and boiling. Samples were normalized for protein content using the Bradford assay using bovine serum albumin as a control.
5
Western Blot Analysis of EMT Markers
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RIPA cell lysis buffer (Beyotime Institute of Biotechnology) was used to obtain cell lysates. Proteins were quantified using bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) then 20 µg protein were loaded per lane and separated via SDS-PAGE on a 10% gel. Separated proteins were then transferred to polyvinylidene difluoride membranes. Following blocking with 5% fat-free milk in PBS for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (cat. no. MB001; 1:2,000; Bioworld Technology, Inc.), NRP-1 (cat. no. ab81321; 1:1,000; Abcam), N-cadherin (cat. no. sc-59987; 1:1,000; Santa Cruz Technology, Inc.), vimentin (cat. no. BS1855; 1:1,000; Bioworld Technology, Inc.), E-cadherin (cat. no. sc-71007; 1:1,000; Santa Cruz Technology, Inc.) and β-catenin (cat. no. ab32572; 1:1,000; Abcam) at 4°C overnight. Membranes were then incubated with horseradish peroxidase-conjugated goat anti-mouse (cat. no. BS12478) or anti-rabbit (cat. no. BS13278; both 1:5,000; Bioworld Technology, Inc.) secondary antibodies for 1 h at room temperature. Following washing, the proteins of interest were visualized by enhanced chemiluminescence Plus Western Blotting Substrate (Thermo Fisher Scientific, Inc.) and ChemiDoc Gel Imaging System (Bio-Rad Laboratories, Inc.).
6
Western Blot Analysis of Protein Expression
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Cells were digested with RIPA cell lysis buffer (Beyotime Biotechnology, Shanghai, China) with protease and phosphatase inhibitor cocktails (MCE, Shanghai, China) and were collected using a cell scraper. Total protein was extracted and quantified using the bicinchoninic acid method (Thermo Fisher Scientific). Proteins (20–30 μg) were resolved using 10% PAGE (Bio-Rad, Hercules, CA, USA), transferred to a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA) at 25 V for 30 min, and blocked for 1 h in 10% nonfat milk in 1× TBS/0.1% (v/v) Tween 20 at room temperature. Primary antibodies (GAPDH, 1:1,000, #5174; Cell Signaling Technology; β-actin, 1:1,000, #4970; Cell Signaling Technology; P1-HNF4A, 1:1,000, ab41898; Abcam; P2-HNF4A, 1:1,000, PP-H6939-00; R & D Systems; and CCL15, 1:1,000, ab219388; Abcam) were added and incubated overnight at 4 °C. Secondary antibodies (goat anti-rabbit IgG, HRP #7074 or goat anti-mouse IgG, HRP, 1:2,000, #7076; Cell Signaling Technology) were incubated at room temperature for 1 h. Signals were detected using Western Lumax Light Sirius HRP substrate reagent (ZETA-Life, San Francisco, CA, USA). All data were normalized to human β-actin or GAPDH. The bands were scanned using a ChemiDocXRS + Imaging System (Bio-Rad).
7
Western Blotting Using RIPA Lysis Buffer
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All of protein extraction use RIPA cell lysis buffer (p0013B) from Beyotime Biotechnology according to the manufacturer's instructions. Protein samples (30-50 μg) were separated by SDS-PAGE, transferred onto PVDF membranes, and blocked with 5% milk. Primary antibodies were diluted with 5% BSA and 4°C overnight. Following incubation, the appropriate secondary antibodies were used from Proteintech (SA00001-2). Finally, chemiluminescence was visualized with Western Bright ECL HRP substrate (Advansta, USA). Equal loading of protein was verified by immunoblotting with anti-β-tubulin or anti-GAPDH antibody.
8
Western Blot Analysis of Exosomal Markers
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Cells were washed separately with ice-cold PBS, gently scraped with a cell spatula, and lysed using the radioimmunoprecipitation assay (RIPA) cell lysis buffer (Beyotime, Shanghai, China). Quantification was performed using the BCA protein concentration assay kit (Beyotime, Shanghai, China). Total protein samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking in 5% nonfat milk, the membranes were probed with antibodies against TRIM62 (ab102012; Abcam), c-Myc (ab32072), TSG101 (ab125011), CD9 (ab92726), CD63 (ab216130), or GAPDH (#5174; CST). The membranes were washed, probed with secondary antibodies (A0208, A0216; Beyotime, Shanghai, China), and then visualized using an ECL kit (Bio-Rad). Results were analyzed using the Image-Pro6.0 software.
9
Protein Expression Analysis of Breast Cancer
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Radioimmunoprecipitation assay (RIPA) cell lysis buffer (Beyotime Biotechnology, Shanghai, China) was used to extract breast cancer and adjacent normal tissues or cells. A bicinchoninic acid (BCA) kit (20201ES76; Yeasen Company, Shanghai, China) was utilized to determine protein concentration. After separated by PAGE, the protein was transferred onto the polyvinylidene fluoride (PVDF) membrane. Nonspecific binding was blocked by addition of 5% BSA at room temperature for 1 h. Subsequently, the protein was incubated with primary antibody rabbit anti-human CDX2 (ab88129, 1:2,000), HOXA5 (ab140636, 1:2,000), E-cadherin (ab15148, 1:500), vimentin (ab137321, 1:2,000), N-cadherin (ab18203, 1:100), Slug (ab106077, 1:2,000), Snail1 (ab82846, 1:300), Twist1 (ab50581, 1:2,000), zinc finger E-box-binding homeobox (ZEB)1 (ab124512, 1:3,000), ZEB2 (ab138222, 1:800), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, 1:5,000) at 4°C overnight. After rinsing with Tris-buffered saline with Tween-20 (TBST), three times (5 min each), the protein was incubated with HRP-labeled goat anti-rabbit IgG (ab205718, 1:20,000) at room temperature for 1 h and visualized by developing liquid. The protein quantitative analysis was conducted using ImageJ 1.48u software (National Institutes of Health, Bethesda, MD, USA) according to the gray value ratio of target band to that of GAPDH band.
10
Western Blot Analysis of Proteins in MDA-MB-231 Cells
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Total proteins in MDA-MB-231 cells were extracted by ice-cold RIPA cell lysis buffer (Beyotime, Shanghai, China). Total proteins (30 μg) of each sample were equally loaded onto SDS-PAGE electrophoresed gels and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% (w/v) skim milk at room temperature for 1 h, the membranes were incubated with primary antibody in Supplementary Methods, followed by incubation with secondary antibodies (1:2000) for 1 h at room temperature. Proteins on the PVDF membrane were visualized using chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). The intensities of the bands were corrected with respect to that of β-Actin. All the experiments were repeated three or more times34 (link),40 (link).
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