The protein was extracted from frozen liver tissue. Cytoplasmic proteins and nuclear proteins were fractionted using a CelLytic™ NuCLEAR™ Extraction kit (Sigma-Aldrich Co. LLC, Beijing, China) (26 (link)). The protein concentration was measured using the Bradford assay. Subsequently, 50 µg protein samples were heated for 10 min at 98°C and subjected to electrophoresis on a 10% SDS-PAGE gel to separate the proteins. The separated proteins were transferred to nitrocellulose membranes (Bio-Trace, USA) and blocked in 5% nonfat milk powder for 2 h at 20°C. After blocking, the protein was incubated overnight at 4°C with anti-keap1 antibody (1:1,500, Abcam) and anti-Nrf2 antibody (1:1,000, Abcam). After washing three times, the corresponding HRP conjugated secondary antibodies were incubated at 4°C. The anti-GADPH antibody (1:1,000, Abcam) and lamin B (1:1,000, Abcam) was used served as loading controls of cytoplasmic and nuclear fractions, respectively. Finally, blots were washed and detected by enhanced chemiluminescence (ECL) using the LumiGlo substrate (Pierce, USA) and Clarity Western ECL Substrate (BioRad, USA).
Anti keap1 antibody
Manufactured by Abcam
Anti-keap1 antibody is a laboratory reagent used for the detection and study of the Keap1 protein in various biological samples. Keap1 is a key regulator of the Nrf2 transcription factor, which plays a central role in the cellular response to oxidative stress. The anti-keap1 antibody can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to investigate the expression, localization, and interactions of the Keap1 protein.
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Lab products found in correlation
Anti nrf2 antibody, by Abcam (1 mentions)
Lamin b, by Abcam (1 mentions)
Anti gadph antibody, by Abcam (1 mentions)
Cellytic nuclear extraction kit, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
β actin 13e5 rabbit mab, by Cell Signaling Technology (1 mentions)
Bca analysis, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
3 protocols using anti keap1 antibody
1
Fractionation and Western Blot Analysis
2
Protein Extraction and Western Blot Analysis
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Pellets from 1 × 106 cells were collected and resuspended with 100 μL RIPA Buffer (Beyotime, Cat: #P0013J). The samples were incubated on ice for 30 min and centrifuged at 15000×g for 15 min at 4 °C. The supernatant was collected, and the protein concentration was measured by BCA analysis (Thermo Scientific, Cat: #23225). Approximately 50 μg of total protein was loaded for western blotting. Antibodies used in this study were HRP AffiniPure Goat anti-Rabbit IgG (H + L) secondary antibody (EARTHOX, Cat: 620822, 1:2000), β-Actin (13E5) Rabbit mAb (Cell Signaling Technology, Cat: #4970, 1:1000), NRF2 (D1Z9C) Rabbit mAb (Cell Signaling Technology, Cat: #12721, 1:1000), Anti-Keap1 antibody (abcam, Cat: ab227828, 1:1000), Anti-IRG1 antibody (abcam, Cat: ab222411, 1:1000). Representative images were obtained using the ChemiDoc Touch Imaging System (Bio-Rad).
3
Keap1-Nrf2 Signaling Pathway in mDCs
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As described [24 (link)], following co-culture with tHSC, 500 μg of total cell lysates of mDCs were first pre-cleared by using A/G Sepharose (“Beads”, Sigma-Aldrich). Thereafter, an anti-Keap1 antibody (Abcam) was included in the pre-cleared lysates overnight, followed by adding back the protein A/G Sepharose for 2h. Keap1-immunoprecipitated Nrf2 was then tested via Western blotting.
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