Protein was extracted from HT29 cells using ice-cold RIPA buffer (Elabscience Biotechnology, Inc.) and determined by using a BCA protein assay kit (Phygene). Protein samples were subjected to separation via 10% SDS-PAGE (DetaiBio Tech) and transferred onto PVDF membranes (Roche Diagnostics), after which time the membranes were blocked with 5% non-fat milk for 2 h at room temperature. The membranes were incubated at 4˚C overnight with the following primary antibodies (all Abcam): Nrf2 (1:1,000, cat. no. ab137550), heme oxygenase-1 (HO-1; 1:2,000, cat. no. ab52947), NADPH dehydrogenase quinone 1 (NQO1; 1:10,000, cat. no. ab80588), GPX4 (1:1,000, cat. no. ab125066), ferritin heavy chain 1 (FTH1; 1:1,000, cat. no. ab183781), transferrin (1:1,000, cat. no. ab277635) and GAPDH (1:1,000, cat. no. ab8245). Following primary antibody incubation, the membranes were incubated with HRP-conjugated secondary antibodies (goat anti-mouse IgG H&L, 1:2,000, cat. no. ab6789; or goat anti-rabbit IgG H&L, 1:2,000, cat. no. ab6721) for 2 h at room temperature. An ECL Western Blotting Substrate kit (AmyJet Scientific, Inc.) was applied to visualize protein bands. The resultant images were analyzed using ImageJ software (version 1.8.0; National Institutes of Health) and the quantification of each group was performed in triplicate.
Ripa buffer
Manufactured by Elabscience
Sourced in China
RIPA buffer is a cell lysis and protein extraction solution used in cell biology and molecular biology applications. It is designed to solubilize and extract proteins from cells and tissues. The buffer contains a combination of non-ionic and ionic detergents, as well as other components that help to maintain protein structure and activity.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Phygene (2 mentions)
Ecl western blotting substrate kit, by AmyJet Scientific (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Pvdf membrane, by Solarbio (1 mentions)
β actin, by Elabscience (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
Chemiluminescence imaging system, by Bio-Rad (1 mentions)
5 protocols using ripa buffer
1
Protein Expression Analysis in HT29 Cells
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from HPMECs using ice-cold radioimmunoprecipitation assay (RIPA) buffer (Elabscience Biotechnology, Inc.). Protein concentrations were then measured using a bicinchoninic acid (BCA) Protein Assay kit (Phygene) in accordance with the manufacturer’s protocol. Protein samples were electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked for 2 h. Subsequently, membranes were incubated with appropriate primary antibodies (all primary antibodies concentration diluted 1000 times) at 4°C overnight. Horseradish peroxidase (HRP)-conjugated secondary antibodies were then added and incubated at room temperature. After washing with PBS, an Ultra High Sensitivity Enhanced Chemiluminescence kit (GlpBio Technology) was prepared and adopted to detect protein bands. Finally, the blotting film was placed in a luminescent imager for photography with ImageJ software (National Institutes of Health).
3
Nanoparticle-Induced Protein Expression
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The cells were grouped and treated as in Section 2.5. After 24 h of treatment with nanoparticles, total proteins were extracted using RIPA buffer (Elabscience Biotechnology Co., Ltd.). The proteins were separated using 10% gel (Epizyme, shanghai) and then transferred to PVDF membranes (Solarbio, Beijing). After blocking with fast blocking western (Solarbio, Beijing) for 10 min at room temperature, PVDF membranes were incubated with primary antibodies (CST, 1:1,000) overnight at 4°C. β-Actin (Elabscience, 1:1,000) was used as an internal reference. The membranes were then incubated with the secondary antibody HRP goat anti-rabbit IgG (Elabscience, 1:5,000) for 1 h. Finally, each group of proteins was detected using electrochemiluminescent ECL reagents. The protein bands were quantified using Image software.
4
Western Blot Protein Quantification
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Ice-cold RIPA buffer (Elabscience, China), containing protease inhibitor (Elabscience, China), was used to extract cell protein, and the protein concentration was detected by the bicinchonininc acid (BCA) method. Protein (30 μg) was loaded into each well on a 10% or 12% SDS–PAGE gel for electrophoresis, and the protein was then transferred to a PVDF membrane. After blocking with 5% nonfat milk for 1 h at room temperature, the PVDF membrane was incubated with primary antibodies at 4°C overnight. After washing with TBST, the membrane was incubated with HRP-conjugated secondary antibodies in blocking buffer at room temperature for 1 h. After three 5 min washes with TBST, the protein bands were visualized using FluorChem Q (ProteinSimple, San Jose, CA, USA) and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA). The GAPDH signal was used to calculate the relative expression of the target protein.
5
Western Blot Analysis of Protein Expression
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Western blot was used to analyze protein expression as described previously [36 (link)]. In brief, total proteins of cells or sEVs were extracted with RIPA buffer (Elabscience) and determined by BCA assay according to the instructions. Equal proteins were loaded on SDS-PAGE gels and transferred to nitrocellulose membranes, then the membranes were blocked with 5% fat-free milk and subsequently incubated with primary antibodies at 4 ℃ overnight. After incubation with peroxidase-conjugated secondary antibodies, target bands were visualized under Chemiluminescence Imaging System (Bio-Rad, Hercules, CA, USA) and the expression levels of the target proteins were analyzed by ImageJ Software (Bio-Rad, USA).
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