Anti keap1

Manufactured by Abcam

Sourced in United States

Anti-Keap1 is a primary antibody that binds to the Keap1 protein. Keap1 is a key regulator of the Nrf2 transcription factor, which plays a crucial role in the cellular response to oxidative stress. The Anti-Keap1 antibody can be used in various research applications to study the Keap1-Nrf2 pathway.

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Lab products found in correlation

13 protocols using anti keap1

Western Blot Analysis of Protein Oxidation

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Protein lysates (20–40 µg) were separated by electrophoresis on a 4–20% SDS polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After blocking with 5–10% BSA, membranes were incubated with primary antibodies for 1 h at room temperature or overnight at 4 °C. The following primary antibodies were used in this study: anti-dinitrophenol (#S7150, as part of the OxyBlot Protein Oxidation Detection Kit, EMD Millipore, 1:150), anti-SQSTM1/p62 (#7695, Cell Signaling, 1:500), anti-Nrf2 (#D1Z9C, Cell Signaling, 1:1000), anti-Keap1 (#ab150654, Abcam, 1:1000), anti-TOPO II β (#ab125297, Abcam, 1:1000), anti-LC3 (#D50G8, Cell Signaling, 1:1000), anti-ubiquitin (#sc-8017, Santa Cruz, 1:250), anti-GCLC (#PA5–44189, Invitrogen, 1:1000), anti-glutathione reductase (#ab16801, Abcam, 1:1000), anti-Bcl2 (#sc-7382 Santa Cruz, 1:1000), anti-GAPDH (#5174, Cell Signaling, 1:2,000), and anti-actin (#sc-1616, Santa Cruz, 1:500). The blots were then incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000) and developed using a chemiluminescence detection system (Santa Cruz Biotechnology or LiCor Premium substrate). Band intensity was determined by densitometry with NIH Image J software (v1.48h3) using mean gray value minus background.
In some experiments, cells were treated with bafilomycin A1 (50 nM, #S1413, SellectChem) at 37 °C prior to lysis.

Pennington S.M., Klutho P.R., Xie L., Broadhurst K., Koval O.M., McCormick M.L., Spitz D.R, & Grumbach I.M. (2018). Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription. Redox Biology, 16, 401-413.

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Protein Expression Analysis in Intestinal Cells

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The protein expression levels of BRG1, Nu-Nrf2, HO-1, Keap-1 and caspase-3 were measured by Western blotting. Generally, total proteins were extracted from NCM460 cells and mouse IECs using RIPA buffer containing protease inhibitors (Roche, Basel, Switzerland), and nuclear proteins were extracted from cells with a nuclear protein extraction kit (Beyotime, Shanghai, China). Then, the protein concentrations were detected with a BCA protein assay kit (Beyotime, Shanghai, China). 10% SDS-PAGE was used to separate equal amount of proteins (40 μg) and transferred onto a PVDF membrane (Millipore, Boston, MA, USA) by electroblotting. The membrane was then blocked with 5% BSA and incubated with the appropriate primary antibodies, including anti-BRG1 (1:1000, Abcam, Cambridge, U.K), anti-Nrf2 (1:1000, CST, Danvers, MA, USA), anti-HO-1 (1:1000, CST, Danvers, MA, USA), anti-Keap-1 (1:1000, Abcam, Cambridge, U.K.), anti-β-actin (1:1000, Beyotime, Shanghai, China), and anti-Lamin B2 (1:1000, CST, Danvers, MA, USA) at 4°C overnight. Afterward, the membrane was incubated with secondary antibodies (1:5000; Beyotime, Shanghai, China) conjugated with peroxidase for 1 h at room temperature. The proteins were detected by an enhanced chemiluminescence (ECL) kit (Beyotime, Shanghai, China). Protein expression was analyzed using Image J software.

Wang X.J., Zhang D., Yang Y.T., Li X.Y., Li H.N., Zhang X.P., Long J.Y., Lu Y.Q., Liu L., Yang G., Liu J., Hong J., Wu H.G, & Ma X.P. (2023). Suppression of microRNA-222-3p ameliorates ulcerative colitis and colitis-associated colorectal cancer to protect against oxidative stress via targeting BRG1 to activate Nrf2/HO-1 signaling pathway. Frontiers in Immunology, 14, 1089809.

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Cellular Antioxidant Mechanisms Explored

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EGCG (purity ≥ 98%) was provided by Teaturn Bio-pharmaceutical Co., Ltd. (Wuxi, China). Anti-TFR1, FPN, FTH/L, NRF2, pNRF2, GPX4, P62, and HO-1 were purchased from Abcam; anti-KEAP1, SLC3A2, and SLC7A11 were purchased from Cell Signaling Technology; and β-actin was purchased from Santa Cruz biotechnology.

Yang C., Wu A., Tan L., Tang D., Chen W., Lai X., Gu K., Chen J., Chen D, & Tang Q. (2023). Epigallocatechin-3-Gallate Alleviates Liver Oxidative Damage Caused by Iron Overload in Mice through Inhibiting Ferroptosis. Nutrients, 15(8), 1993.

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Aortic Protein Expression Analysis

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Aortas were frozen in liquid nitrogen and homogenized in a lysis buffer [50 mM Tris/HCl, 150 mM NaCl, 1% Non-idet P40, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 μg/mL leupeptin, 1 μg/mL pepstatin, 1 μg/mL aprotinin, 1 mM sodium orthovanadate, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mM sodium fluoride]. The tissue extracts were centrifuged, and total protein content was quantified using the Bradford method (Bradford, 1976 (link)). Proteins (40 μg) were separated by electrophoresis on 10% polyacrylamide gel, and transferred on to nitrocellulose membranes. Non-specific binding sites were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Tween 20 (for 1 h at 24°C). Membranes were incubated with antibodies (at the indicated dilutions) overnight at 4°C. Antibodies were used as follows: anti-keap1 (1:1,000 dilution; Abcam), anti-NOX1 (1:1,000 dilution; Cell Signaling), anti-NOX4 (1:1,000 dilution; Cell Signaling), anti-βactin (1:1,0,000 dilution; Sigma), and anti-GAPDH (1:10,000 dilution; Sigma). After incubation with secondary antibodies, signals were obtained by chemiluminescence, visualized by autoradiography and quantified densitometrically.

Costa R.M., Alves-Lopes R., Alves J.V., Servian C.P., Mestriner F.L., Carneiro F.S., Lobato N.D, & Tostes R.C. (2022). Testosterone Contributes to Vascular Dysfunction in Young Mice Fed a High Fat Diet by Promoting Nuclear Factor E2–Related Factor 2 Downregulation and Oxidative Stress. Frontiers in Physiology, 13, 837603.

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Western Blot Analysis of Cellular Proteins

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Cell lysates were prepared in M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) with Mini protease and Halt phosphatase inhibitor cocktails (Thermo Scientific) and protein concentration was determined using the BCA assay (Thermo Scientific). 25 μg of total cellular protein was separated on SDS/PAGE gels and transferred to nitrocellulose membrane that was blocked for 1 hour at room temperature in 5% nonfat milk. Primary antibodies were added overnight at 4°C with gentle shaking. The primary antibodies used were: anti-TP53 (Sigma), anti-CDKN1A (Cell Signaling), anti-NRF2 (Abcam), anti-KEAP1 (Abcam), anti-ubiquitin (Cell Signaling), anti-HSP70 (Enzo), anti-HSP90 (Cell Signaling), anti-pAKTThr308 (Cell Signaling), anti-pAKTSer473 (Cell Signaling) and anti-AKT (Cell Signaling). After incubation with the appropriate secondary antibody, signals were detected using ECL Western Blotting Substrate (Thermo Scientific). Equal protein loading in each lane was confirmed with β-actin antibody (Sigma). Densitometry analysis was performed using the freely available image-processing program ImageJ (NIH).

Hyter S., Hirst J., Pathak H., Pessetto Z.Y., Koestler D.C., Raghavan R., Pei D, & Godwin A.K. (2017). Developing a genetic signature to predict drug response in ovarian cancer. Oncotarget, 9(19), 14828-14848.

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Western Blot Analysis of Endothelial Protein Expression

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Protein expression of p38 MAPK (total and phosphorylated), Keap1, Bach1, and beta-actin was determined by Western blot analysis in cultured endothelial cells and isolated aortas. Samples were homogenized in lysis buffer, and proteins were kept frozen in −80 °C until use. Proteins (30 μg) were separated by electrophoresis on 10% or 12% polyacrylamide gels, transferred to 0.22 μm nitrocellulose membranes, and blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) and 0.1% Tween 20 for 1 h. Primary antibodies were incubated overnight at 4 °C, as follows: anti-phospho p38 (phospho Thr180 + Tyr182) (1:1000; Cell Signaling 4511), anti-p38 (1:1000; Cell Signaling 9212), anti-Keap1 (1:1000; Abcam 66620), anti-Bach1 (1:1000; sc-271211), and anti-β-actin-peroxidase (1:10,000 dilution; Sigma-Aldrich). Protein bands were detected by chemiluminescence reaction (Luminata Forte, WBLUF0100, Merck-Millipore, Watford, UK), and the intensity of the bands was evaluated by densitometric analysis using ImageQuant software.

Rodrigues D., Costa T.J., Silva J.F., Neto J.T., Alves J.V., Fedoce A.G., Costa R.M, & Tostes R.C. (2021). Aldosterone Negatively Regulates Nrf2 Activity: An Additional Mechanism Contributing to Oxidative Stress and Vascular Dysfunction by Aldosterone. International Journal of Molecular Sciences, 22(11), 6154.

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Western Blot Analysis of Apoptosis and Nrf2 Pathway

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Proteins were extracted from cell lysates, using RIPA lysis buffer, and the protein concentration was measured using the Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology), following the manufacturer’s recommendations. Proteins were denatured by heat, and then separated by SDS-PAGE electrophoresis, and finally transferred to PVDF membranes. The membranes were blocked using 1% bovine serum albumin solution for 1 h at room temperature, and then incubated with primary antibodies, including anti-cleaved caspase-3 (1:1000; Abcam), anti-Keap1 (1:1000; Abcam), anti-Nrf2 (1:1000; Abcam), anti-p-Nrf2 (1:1000; Abcam), anti-heme oxygenase-1 (1:1000; Abcam), anti-NAD(P)H quinone dehydrogenase 1 (1:1000; Abcam) and anti-β-actin (1:1000; Zhongshan Jinqiao Biotechnology, Beijing, China) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000; Zhongshan Jinqiao Biotechnology, Beijing, China) at room temperature for 30 min. Protein bands were detected using a Immun-Star HRP kit (Bio-Rad), following the manufacturer’s protocols. Relative densitometry was analyzed using the Image J2x analysis software (National Institutes of Health, USA).

Shi X., Tao G., Ji L, & Tian G. (2020). Sappanone A Protects Against Myocardial Ischemia Reperfusion Injury by Modulation of Nrf2. Drug Design, Development and Therapy, 14, 61-71.

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Protein Extraction and Western Blot Analysis

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According to the manufacturer’s instructions, the protein extracts of nucleus and cytoplasm (three mice in each group) were prepared using nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Shanghai, China). The total protein was assayed using BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins (50 μg) were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After blocked with 5% nonfat milk for 1 h, the membranes were incubated overnight at 4 °C with the primary antibodies for cytosolic fractions: rabbit anti-TH antibody (1:1000, Millipore Corporation), rabbit anti-phospho-PI3K (1:1000, Cell Signaling Technology), rabbit anti-PI3K (1:1000, Cell Signaling), rabbit anti-phospho-AKT (1:1000, Cell Signaling), rabbit anti-AKT (1:1000; Cell Signaling), anti-GCLC (1:1000, Abcam), anti-GCLM (1:2000, Abcam), anti-Heme Oxygenase 1 (1:2000, Abcam), anti-Keap1 (1 μg/ml, Abcam), rabbit anti-β-Actin (1:1000; Cell Signaling), and the primary antibodies for nuclear fractions: anti-Lamin B1 (0.1 μg/ml, Abcam), anti-Nrf2 (1:1000, Abcam). The membranes were incubated with the horseradish peroxidase-labeled secondary antibody (1:2000). Bands were investigated using the ECL detection reagents (Beyotime Institute of Biotechnology, Shanghai, China).

Wang T., Li C., Han B., Wang Z., Meng X., Zhang L., He J, & Fu F. (2020). Neuroprotective effects of Danshensu on rotenone-induced Parkinson’s disease models in vitro and in vivo. BMC Complementary Medicine and Therapies, 20, 20.

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Western Blot Analysis of Testicular Proteins

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Testicular protein (50 μg per well) was separated by 12% SDS-PAGE gels; then, the protein was transferred using the polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 4% skim milk. Immunoblotting was performed with the first antibody then the secondary antibody (1:5000) (ZSGB-BIO, Beijing, China). First antibody: anti-Nrf2 (ab31163, 1:1500 dilution; Abcam, London, Britain); anti-keap1 (ab119403, 1:1500 dilution; Abcam); anti-LC3B (ab63817, 1:1000 dilution; Abcam); anti-SQSTM1/P62 (#23214, 1:1000 dilution; Cell Signaling Technology, Danvers, MA, country); and anti-B ECLin1 (#3738, 1:1000 dilution; Cell Signaling Technology). Signals were detected by chemiluminescence (ECL, Thermo Fisher Scientific, Waltham, MA, USA). Each experiment was repeated at least three times.

Huo L., Su Y., Xu G., Zhai L, & Zhao J. (2019). Sulforaphane Protects the Male Reproductive System of Mice from Obesity-Induced Damage: Involvement of Oxidative Stress and Autophagy. International Journal of Environmental Research and Public Health, 16(19), 3759.

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Western Blot Analysis of Ubiquitination

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Proteins were resolved by SDS-PAGE (sodium-dodecyl sulfate poly-acrylamide gel electrophoresis) and were probed with the following antibodies: anti-Nrf2 (Abcam, Cambridge, MA; cat no: 62352), anti-GFP (ThermoFisher, Waltham, MA; cat no: A11122), anti-FLAG (Sigma Aldrich, St. Louis, MO; cat no: F1804 and F3165), anti-K48 ubiquitin specific (Cell Signaling Technology, Boston, MA; cat no: 8081S), anti-K63 ubiquitin specific (Abcam, Cambridge, MA; cat no: ab179434), anti-HA (Vanderbilt University Molecular Core, Nashville, TN), anti-RNF111 (Abnova, Taipei, Taiwan; cat no: H00054778), anti-β-actin-peroxidase (Sigma Aldrich, St. Louis, MO; cat no: A3854), anti-LDH (Epitomics, Burlingame, CA; cat no: 1980–1), anti-AKAP95 (Millipore, Burlington, MA; cat no: 06–417), and anti-Keap1 (Abcam, Cambridge, MA; cat no: ab31973) antibodies.

McIntosh D.J., Walters T.S., Arinze I.J, & Davis J.S. (2018). Arkadia (RING Finger Protein 111) Mediates Sumoylation-Dependent Stabilization of Nrf2 Through K48-Linked Ubiquitination. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 46(1), 418-430.

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