Total protein was isolated from dermal tissues and fibroblasts by using RIPA buffer (10X) (Cell Signaling) and quantified by a BCA assay kit (Sigma‐Aldrich). After separating the total protein via sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, proteins were transferred onto polyvinylidene difluoride membranes. Then, membranes were blocked for 2 h and subsequently incubated with primary antibodies overnight. The next day, membranes were rinsed three times and further incubated with horseradish peroxidase‐conjugated secondary antibody for 2 h. Finally, protein bands were visualized with an ECL detection reagent (Sigma‐Aldrich). Antibodies used in this study were all supplied by Abcam, and the corresponding information was listed as followed: α‐SMA (Cat. no. ab5894; 1 µg/ml), collagen I (Cat. no. ab138492; 1/2000 dilution), phosphor‐ERK1/2 (p‐ERK1/2; Cat. no. ab278538; 0.1 µg/ml), ERK1/2 (T‐ERK1/2; Cat. no. ab184699; 1/10,000 dilution), phosphor‐Smad2 (p‐Smad2 (S467); Cat. no. ab280888; 1/1000 dilution), Smad2 (T‐Smad2; Cat. no. ab40855; 1/5000 dilution), phosphor‐Smad3 (p‐Smad3 (S423 + S425); Cat. no. ab52903; 1/2000 dilution), Smad3 (T‐Smad3; Cat. no. ab40854; 1/5000 dilution), and β‐actin (Cat. no. ab8226; 1 µg/ml).
Ripa buffer 10x
Manufactured by Cell Signaling Technology
Sourced in United States
RIPA buffer (10X) is a concentrated solution used for cell lysis and protein extraction. It contains a combination of detergents and other components that help solubilize cellular proteins and disrupt cell membranes. The buffer is designed to maintain the native structure and function of proteins during the extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Bca assay kit, by Merck Group (2 mentions)
Ecl detection reagent, by Merck Group (2 mentions)
Phosstop and complete mini, by Roche (2 mentions)
Lds loading buffer, by Thermo Fisher Scientific (2 mentions)
Transfer buffer, by Bio-Rad (1 mentions)
Vivaspin turbo 15, by Sartorius (1 mentions)
Superdex 30 increase sec columns, by GE Healthcare (1 mentions)
Tbst buffer, by Bio-Rad (1 mentions)
Phosphor p65, by Cell Signaling Technology (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Ecl substrate, by Bio-Rad (1 mentions)
Trypsin edta solution, by GE Healthcare (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Vivaspin 15r, by Sartorius (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
7 protocols using ripa buffer 10x
1
Western Blot Analysis of Dermal Protein
2
Western Blot Analysis of Protein Samples
3
Rhizopus microspores Fermentation of Quinoa
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C. formosanum grains were obtained from Quinoa Green Biotech Co., Ltd (Taichung, Taiwan). Rhizopus microspores var oligosporus BCRC 31996 and murine alveolar macrophage MH-S cell line were sourced from Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan). Agar, potato dextrose broth, and peptone, which constitute the medium, were acquired from BioShop Canada Inc (Ontario, Canada). Superdex 30 Increase SEC columns, Sephadex G-25 columns resin, Penicillin/streptomycin and trypsin EDTA solution, used for protein purification and cell culture, were procured from GE Healthcare Life Science (Logan, Utah, USA). Vivaspin 15R and Vivaspin Turbo 15 were obtained from Sartorius Stedim Biotech GmbH (Goettingen, Germany). GIBCO Life Technologies (Grand Island, USA) supplied the cell culture mediums and fetal bovine serum. Immunoblot-related materials, namely RIPA buffer (10X), phosphor-p65, beta-actin, and p65 antibody, were purchased from Cell Signaling Technology (Beverly, MA, USA). TBST Buffer, Transfer Buffer, ECL substrate, and protein assay dye reagent were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Goat anti-rabbit IgG antibodies (HRP) were acquired from Genetex (Irvine, CA, USA). All the chemicals utilized in the research were of analytical grade and were procured from Merck (Burlington, MA, USA).
4
Protein Expression Analysis by Western Blotting
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Myocytes were rinsed with PBS and scraped from the Petri dishes in 1X RIPA lysis buffer (RIPA buffer 10X, Cell Signaling) supplemented with 0.1% SDS, protease inhibitor cocktail III (Calbiochem) and phosphatase inhibitor cocktail (Sigma). DC Protein Assay from BioRad was used to determine total protein. Samples were treated with β-mercaptoethanol and heated to 95°C for 5 min. Proteins were separated by SDS/PAGE and transferred to PVDF membrane (BioRad). Blots containing either whole cell lysates or fractionated cells were probed with primary antibodies: eIF4A3 1:500 (antibody a) or 1:5000 (antibody b); all other antibodies at 1:1000 except actin (1:5000). Horseradish peroxidase-conjugated secondary antibodies anti-mouse, anti-rabbit or anti-goat (Thermo-Scientific, Life Technology) were used to visualize proteins by enhanced chemiluminescence (ECL) solutions of various sensitivity (Thermo Scientific West Pico & Femto; BioRad Clarity), depending on the strength of the chemiluminescent signal. The bands corresponding to the various proteins were detected on ImageQuant LAS 4000 (GE Healthcare) and quantified by densitometry using ImageJ software. Protein bands were standardized to total protein as previously described [13 (link)].
5
Western Blot Protocol for Protein Analysis
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Human tissue and cellular preparations were harvested in radio immunoprecipitation buffer (RIPA) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycorophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin (RIPA Buffer, 10x, CST#9806, Cell Signaling, Danvers, MA, USA) and one tablet each of PhosStop and Complete Mini (Roche, Indianapolis, IN, USA). Insoluble materials were removed by centrifugation at 14 000 r.p.m. for 20 min at 4 °C. Protein concentration was determined using the bicinchronic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA). Samples were prepared with 10–30 μg of protein in RIPA buffer with 4 × LDS loading buffer (Cat#LP0001, Life Technologies). Samples were electrophoresed on SDS–PAGE gels, transferred to PVDF membranes, probed with primary antibodies overnight, then secondary antibodies for 1 h at room temperature (Supplementary Table S3 ). Membranes were developed using Clarity Western ECL substrate (Cat#10026385 Rev A, Bio-Rad) and radiographic film.
6
Western Blot Analysis of Stemness Markers
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Western blot was performed as previously described [44 (link)]. In brief, after isolating total protein from tissues and cells with RIPA buffer (10X) (Cell Signaling, MA, USA), protein quantification was performed with a BCA assay kit (Sigma-Aldrich, MO, USA). The total protein was separated by SDS-PAGE gel, and subsequently transferred onto PVDF membranes. After blocking with skim milk, membranes were incubated with primary antibodies specific for USP36, CD133, CD44, Nanog, Oct-4, c-Myc, and GADPH overnight. Next, membranes were washed thrice prior to 2 h incubation with HRP-conjugated secondary antibody. At last, an ECL detection reagent (Sigma-Aldrich, MO, USA) was used to visualize protein bands. The information on antibodies used in this study was listed in Supplementary Table 1 .
7
Western Blot Protein Extraction Protocol
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Cells were lysed for protein extraction by incubating for 5 min in RIPA buffer (10X, #9806, Cell Signaling) on ice after washing with PBS twice. Cells were then scraped and sonicated briefly, centrifuged for 10 min at 14,000x g in +4°C. Protein concentrations were measured with Bradford or a Nanodrop 2000 instrument. 20–40µg of protein was run into a 10% or 12% polyacrylamide gel (Bio Rad) at 100V for 10 min and then 150V for 50 min. Semi-dry blotting (Thermo Fisher) was conducted transferring the proteins to a PVDF transfer membrane (Merck Millipore) under 36mA for 65 min. Alternatively, proteins were transferred to a nitrocellulose membrane (AmershamTM ProtanTM, GE Healthcare Life Sciences) by wet blotting under 45 V for 15 min, then 120 V for 45 min. 5% milk/TBST was used for blocking the membrane for 1 h at RT followed by incubation with primary antibodies overnight in +4 °C. On the following day, the membrane was washed with TBST and incubated with secondary antibody for 1 h at RT. After washing with TBST, ECL Prime Detection Reagent (GE Healthcare) or ClarityTM Western ECL Substrate (BioRad) was used to detect the bands for imaging with a Chemidoc XRS+ Molecular Imager (Bio-Rad).
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