pOPTN-eGFP (#27052), pcDNA3-HA2-Keap1 (#21556), and pHA-tag (#55182) were purchased from addgene.org . For expressed co-IP assay, HEK293T cells were seeded in six-well plates until 80% confluency and transfected with indicated plasmids at a concentration of 2000 ng per plasmid per well by Lipofectamine 2000 according to the manufacturer’s protocol. Transfected cells were lysed by NP-40 buffer and split into two parts—200μl fractions for input assay and an 800 μl of fractions for co-IP. The input lysates were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) gels and analyzed by immunoblot. The co-IP lysates were incubated with 20 μl of EZview Red Anti-HA Affinity Gel (E6779-1ML, MilliporeSigma) for 24 hours. For endogenous co-IP assay, osteoclasts from 2-month-old Optn+/+ mice were used. The co-IP lysates were precipitated with anti-KEAP1 antibody (sc-514914, Santa Cruz Biotechnology) or normal mouse immunoglobulin G control (#02-6502, Invitrogen) bound to protein G and protein A agarose beads (IP05, MilliporeSigma), respectively, for 24 hours. After the incubation, samples were centrifuged at 5000g for 1 min, and pellets were washed five times with NP-40 buffer. The proteins were lastly resolved on SDS-PAGE gels and analyzed by immunoblot.
Anti keap1 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-Keap1 antibody is a research-use antibody produced by Santa Cruz Biotechnology. It is designed to detect the Keap1 (Kelch-like ECH-associated protein 1) protein, which is a key regulator of the Nrf2 (Nuclear factor erythroid 2-related factor 2) signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti nrf2 antibody, by Santa Cruz Biotechnology (4 mentions)
Ezview red anti ha affinity gel, by Merck Group (1 mentions)
Poptn egfp, by Addgene (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Streptavidin agarose beads, by Merck Group (1 mentions)
Mg132, by Enzo Life Sciences (1 mentions)
Nitrocellulose blotting membrane, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection kit, by iNtRON Biotechnology (1 mentions)
Bca assay kit, by iNtRON Biotechnology (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti nrf2 antibody, by Proteintech (1 mentions)
3 methyladenine, by Merck Group (1 mentions)
Anti c myc antibody, by Merck Group (1 mentions)
Anti β actin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Mln4924, by Active Motif (1 mentions)
Bafilomycin a1, by MedChemExpress (1 mentions)
10 protocols using anti keap1 antibody
1
Co-immunoprecipitation and Immunoblot Analysis
2
Visualizing PD-L1 and KEAP1 in Lung Cancer
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H1299 and H23 cells were seeded in confocal dishes (Biofilm) for 48 hours and then fixed with 4% paraformaldehyde diluted in PBS for 15 minutes at room temperature. After being washed with PBS for three times, cells were incubated with a blocking buffer containing 0.25% triton X-100 for 1 hour, then incubated with anti-PD-L1 antibody (1:100, Proteintech, 17952-1-AP) and anti-KEAP1 antibody (1:100, Santa Cruz, sc-365626) at 4 °C overnight. And then, cells were washed with PBS for three times and incubated with Alexa Fluor 488-conjugated anti-mouse secondary antibody and Alexa Fluor 555-conjugated anti-rabbit secondary antibody for 2 hours at room temperature followed by incubation with DAPI for 5 minutes before being investigated with confocal microscope.
3
Nrf2-KEAP1 Interaction Assay
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Cells were lysed using a lysis buffer (1% NP-40, 150 mM NaCl, 1 mM EDTA, 0.3 µM aprotinin, 1 µM pepstatin, and 1 mM PMSF). The cell lysates (0.7–0.8 mg protein) were incubated with BT-MA (1 μM) at 30 °C for 2 h in the presence or absence of 100 μM TRL or 200 μM rTRL. Streptavidin-agarose beads (20 µL; Millipore, Hayward, CA, USA) were added to the lysates and incubated at 4 °C for 2 h. The beads were washed five times with washing buffer (20 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40). For co-immunoprecipitation, cells pretreated with the proteasome inhibitor MG132 (Enzo Life Sciences, New York, NY, USA) for 2 h were lysed using RIPA buffer. The cell lysates were incubated at 30 °C for 2 h with TRL (100 μM), followed by the addition of the anti-KEAP1 antibody (sc-365626, Santa Cruz Biotechnology) at 4 °C for 2 h, after which protein A/G agarose (20 µL, Santa Cruz Biotechnology) was added at 4 °C for 2 h. The beads were washed five times with radioimmunoprecipitation assay (RIPA) buffer. SDS sample buffer (1×) was added, and the mixture was boiled for 5 m. The precipitated proteins were separated using 7.5% SDS-PAGE gels. Immunoblot analysis was performed with anti-Nrf2 antibody (Santa Cruz Biotechnology, sc-722) and anti-KEAP1 antibody as described above.
4
Nrf2 and Keap1 Protein Expression
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A549 cells were lysed by protein extraction solution about 70 μL (iNtron Biotechnology, Seoungnam, Korea). The protein concentrations of samples were determined through the bicinchoninic acid (BCA) assay kit (iNtron Biotechnology, Korea). A 30 μg of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were electrophoresed for 5 h (60 mA) at room temperature and then transferred onto a nitrocellulose blotting membrane (Bio-rad, Hercules, CA, USA) for overnight. The membrane was blocked with 5% skim milk in tris-buffered saline (TBS) with 0.01% Tween-20 for 1 h and incubated with rabbit polyclonal anti-Nrf2 antibody (1:250 dilution, Santa cruz, sc-13032, Dallas, TX, USA) or goat polyclonal anti-keap1 antibody (1:300 dilution Santa cruz, sc-15246, USA) for overnight. To detect Nrf2, keap1, Horseradish peroxidase-conjugated anti-rabbit IgG or anti-goat IgG (both at 1:5,000 dilution, KPL, Gaitherburg, MD, USA) were incubated as a secondary antibody for 1 h at room temperature. Immunoreactivity was visualized by an enhanced chemiluminescence detection kit (iNtron Biotechnology, Korea).
5
Immunoprecipitation of Keap1 Protein Complex
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As reported previously [4 (link)], following the applied treatments total cell lysates (800 μg proteins per treatment) were pre-cleared and then incubated with an anti-Keap1 antibody (Santa Cruz Biotech, Shanghai, China) overnight. Proteins that were immunoprecipitated with Keap1 were captured by protein IgA/G beads, and were subsequently tested by western blotting. The detailed protocols of isolating mitochondrial fraction lysates and mito-IP were described before [29 (link)].
6
Quantitative Analysis of Nrf2 and Keap1 in Renal Tissue
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For IHC, 3 μm thick paraffin-embedded renal sections were deparaffinized, hydrated and blocked, following incubation with primary antibodies including rabbit anti-Nrf2 antibody (catalog number: 16396-1-AP, Proteintech, Chicago, IL, USA) and anti-Keap1 antibody (catalog number: sc-365626, Santa Cruz, Dallas, TX, USA) overnight at 4 °C. Following diamino-benzidine (DAB) reaction, the sections were then incubated in horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 30 min. Sections were photographed under a light microscope (Olympus CX23, Tokyo, Japan) and quantified with image-pro plus 6.0 software (Media Cybernetics, Rockville, MD, USA) as previously reported [62 ]. After intensity calibration, positive regions were extracted and separated. The positive color segmentation threshold was based on the fixed threshold value of hue, saturation, and intensity (HSI). Images segmentation and area measurement was based on the same HSI profile for all images. Integral optical density (IOD) and the area were measured and the protein expression levels were analyzed by calculating IOD/Area. Four fields of view were randomly selected from each sample.
7
Protein Regulation Analysis Protocol
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MLN4924 (catalog no.A-1139; Activebiochem), 3-methyladenine (Sigma-Aldrich), Bafilomycin A1 (MedChemExpress), Lipofectamine 2000 (Invitrogen), protease inhibitor cocktail (CompleteMini, Roche), phosphatase inhibitor cocktail (PhosStop, Roche).
Immunoblots were probed using the following primary antibodies: anti cullin3 antibody (Cell Signaling Technology), anti-β-actin monoclonal antibody (Santa Cruz), anti-NEDD8 antibody (Cell Signaling Technology), anti-c-Myc antibody (Sigma-Aldrich), anti-p62 antibody (MBL), anti-light chain 3B antibody (LC3B; MBL and Cell Signaling Technology), anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH; Cell Signaling Technology), anti WNK4 and anti pWNK4 antibody (from Oregon Health & Science University), anti Keap1 antibody (Santa Cruz), anti Nrf2 antibody (Santa Cruz). Alkaline phosphatase-conjugated anti-immunoglobulin G (IgG) antibodies (Santa Cruz) were used as the secondary antibodies for immunoblotting.
Immunoblots were probed using the following primary antibodies: anti cullin3 antibody (Cell Signaling Technology), anti-β-actin monoclonal antibody (Santa Cruz), anti-NEDD8 antibody (Cell Signaling Technology), anti-c-Myc antibody (Sigma-Aldrich), anti-p62 antibody (MBL), anti-light chain 3B antibody (LC3B; MBL and Cell Signaling Technology), anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH; Cell Signaling Technology), anti WNK4 and anti pWNK4 antibody (from Oregon Health & Science University), anti Keap1 antibody (Santa Cruz), anti Nrf2 antibody (Santa Cruz). Alkaline phosphatase-conjugated anti-immunoglobulin G (IgG) antibodies (Santa Cruz) were used as the secondary antibodies for immunoblotting.
8
Protein Extraction and Immunoblotting Analysis
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RIPA buffer (Thermo Fisher Scientific) and a Nuclear/Cytosol Extraction Kit (Beyotime, China) were used to extract total and nuclear protein according to the manufacturer's instructions. We used the following antibodies: anti-SQSTM1 antibody (CST, 39749), anti-LC3A/B antibody (CST, 12741), anti-mTOR antibody (CST, 2983), anti-phospho-mTOR antibody (CST, 5536), anti-AMPK antibody (CST, 5831), anti-phospho-AMPK antibody (CST, 50081), anti-Nrf2 antibody (Santa Cruz, sc-365949), anti-keap1 antibody (Santa Cruz, sc-514914), anti-NQO1 antibody (Santa Cruz, sc-32793), anti-HO-1 antibody (Abcam, ab52947), and anti-β-actin antibody (Santa Cruz, sc-47778). The blots were incubated with primary antibodies overnight at 4°C, at which point an anti-rabbit or anti-mouse secondary antibody was added for 1 h. Band intensities were quantified by ImageJ densitometry software.
9
Biotin-mediated Protein Interactome Profiling
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DOPAC, laccase from Rhus vernicifera, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), bis(4-nitrophenyl) phosphate (BNPP) and azide-PEG3-biotin conjugate were obtained from Sigma Aldrich (St. Louis, USA). p-Toluensulfonic acid monohydrate (PTSA), n-butanol, toluene, copper (II) sulfate pentahydrate, protease inhibitor cocktail and Chemi-Lumi One Super were purchased from nacalai tasque (Kyoto, Japan). 2-Propyl-1-ol was obtained from Tokyo Chemical Industry (Tokyo, Japan). Streptavidin, HRP conjugate was purchased from Funakoshi (Tokyo, Japan). Anti-actin antibody, anti-aryl hydrocarbon receptor (AhR) antibody, anti-Keap1 antibody, horseradish peroxidase-linked anti-mouse IgG and horseradish peroxidase-linked anti-goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Streptavidin Mag Sepharose was purchased from GE health care (Little Chalfont, UK). All other chemicals such as benzyl azide were purchased from Wako Pure Chemical Industries (Osaka, Japan).
10
Keap1-Nrf2 Co-immunoprecipitation Assay
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For each treatment, 800 μg of total cell lysates were pre-cleared by the protein A/G Sepharose (Sigma-Aldrich). Thereafter, the lysates were incubated with anti-Keap1 antibody (Santa Cruz Biotechnology) overnight. protein A/G Sepharose (30 µL for each treatment) was then added. Keap1 co-immunoprecipitation with Nrf2 was tested via Western blotting.
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