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Anti keap1 antibody

Manufactured by Proteintech
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The Anti-Keap1 antibody is a laboratory reagent used for the detection and study of 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 antibody can be utilized in various analytical 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

Bca protein assay, by Thermo Fisher Scientific (2 mentions) Proteinase inhibitor cocktail and phosphatase inhibitor, by Santa Cruz Biotechnology (1 mentions) Anti β tubulin antibody, by Cell Signaling Technology (1 mentions) Anti histone h3 antibody, by Cell Signaling Technology (1 mentions) Horseradish peroxidase conjugated anti mouse and anti rabbit secondary antibodies, by Cell Signaling Technology (1 mentions) Chemidoc xrst image system, by Bio-Rad (1 mentions) Anti β actin antibody, by Merck Group (1 mentions) Anti ubiquitin, by Cell Signaling Technology (1 mentions) Anti atg7, by Cell Signaling Technology (1 mentions) Hydrogen peroxide solution h2o2, by Merck Group (1 mentions) Kahweol, by LKT Laboratories (1 mentions) Anti ho 1, by Santa Cruz Biotechnology (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Anti beclin 1, by Cell Signaling Technology (1 mentions) Anti β tubulin, by Cell Signaling Technology (1 mentions) Anti p62, by Abcam (1 mentions) Anti atg5, by Abcam (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Anti nrf2 antibody, by Abcam (1 mentions) Piercetm co immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)

5 protocols using anti keap1 antibody

1

Western Blot Analysis of Cellular Proteins

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Cells were lysed using RIPA buffer in the presence of proteinase inhibitor cocktail and phosphatase inhibitor (Santa Cruz Biotechnology, Dallas, TX). Protein concentration was determined by a BCA Protein Assay (Thermo Scientific) using a FLUOstar OPTIMA microplate reader (BMG). Equal amounts of proteins were loaded and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, followed by Western blot analysis using standard protocols. The primary antibodies were: anti-NRF2 antibody (Millipore), Anti-KEAP1 antibody (Proteintech), anti-cleaved caspase 3 and caspase 3 antibodies (Cell Signaling), anti-cleaved PARP and PARP antibodies (Cell Signaling), anti-histone H3 antibody, anti-β-tubulin antibody (Cell Signaling), and anti-β-actin antibody (Sigma). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Cell Signaling Technology. Immunoreactive protein bands were visualized by enhanced chemiluminescence and images were captured by a ChemiDoc XRST Image System (Bio-Rad).
Peng D., Lu H., Zhu S., Zhou Z., Hu T., Chen Z., Zaika A, & El-Rifai W. (2019). NRF2 antioxidant response protects against acidic bile salts-induced oxidative stress and DNA damage in esophageal cells. Cancer letters, 458, 46-55.
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2

Kahweol-Mediated Nrf2 Activation and Autophagy

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Kahweol was purchased from LKT Laboratories Inc. (St. Paul, MN, USA) and hydrogen peroxide solution (H2O2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-Keap1 antibody was purchased form Proteintech (Rosemont, IL, USA), the anti-Nrf2 (1:2000) antibody was purchased from Thermo Scientific (Waltham, MA, USA), the anti-p62 (1:10000) and Anti-ATG5 (1:2000) antibody antibody was purchased from Abcam (Cambridge, UK), and the anti-HO-1 (1:5000) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-ATG7 (1:2000), anti-β-tubulin (1:10000), anti-cleaved caspase 3 (1:2000), anti-GAPDH, Anti-ubiquitin (1:2000) and anti-Beclin1 (1:2000) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
Seo H.Y., Lee S.H., Lee J.H., Hwang J.S., Kim M.K, & Jang B.K. (2020). Kahweol activates the Nrf2/HO-1 pathway by decreasing Keap1 expression independently of p62 and autophagy pathways. PLoS ONE, 15(10), e0240478.
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3

Nrf2 Immunoprecipitation and Keap1 Analysis

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Co-immunoprecipitation was performed according to the manufacturer’s protocol using the PierceTM CoImmunoprecipitation Kit from Thermo Fisher Scientific (Grand Island, NY, USA) as previously described (Zhang et al., 2019 (link)). Briefly, the samples obtained from rFPA were lysed and extracted followed by centrifugation. After immunoprecipitation was performed using 10 μg of the anti-Nrf-2 antibody (Abcam, Cambridge, MA, USA; ab137550) by 3-hour incubation, protein G sepharose was added to the antibody-coupled resin, incubated overnight at 4 °C, and then centrifuged for 1 minute at 12000g. The resin was washed, and the protein complexes bound to the antibody were eluted. Then, the supernatant was loaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot analyses were performed as previously described to assess the protein levels of Keap-1 in the precipitates using anti-Keap-1 antibody (1: 500; Proteintech Rosemont, IL, USA; 60027-1-Ig).
Ocak P.E., Ocak U., Sherchan P., Gamdzyk M., Tang J, & Zhang J.H. (2020). Overexpression of Mfsd2a Attenuates Blood Brain Barrier Dysfunction via Cav-1/Keap-1/Nrf-2/HO-1 pathway in a rat model of surgical brain injury. Experimental neurology, 326, 113203.
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4

SC-236 Modulated Immunostaining Assay

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Cell infections were performed after 36 h of 5µM of SC-236 treatment as described earlier. After infection, cells were fixed in 4% PFA for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS. Thereafter, cells were blocked for an additional 60 min with the 5% BSA. Cells were stained with anti-psoriasin antibody (Santa Cruz Biotechnology), anti-hBD2 antibody (Santa Cruz Biotechnology), anti-KEAP1 antibody (Proteintech), anti-claudin-1 antibody (Invitrogen), anti-CD86 antibody (BD Biosciences) and anti-TLR4 (BD biosciences) at 1:200 dilutions or anti-NRF2 (Cell Signaling Technologies) at 1:100, or with anti-NOS2 antibody (Santa Cruz Biotechnology) at 1:50 dilution followed by the respective Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 647 conjugated secondary antibody (Life Technologies) at 1:400 dilution and counter-stained using 2.5 µg mL− 1 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Confocal images were acquired on a LSM 700 microscope (Carl Zeiss) using 63× oil immersion objective. All images were processed for intensity quantification by ImageJ software (NIH).
Mohanty S., Lindelauf C., White J.K., Scheffschick A., Ehrenborg E., Demirel I., Brauner H, & Brauner A. (2023). Inhibition of COX-2 signaling favors E. coli during urinary tract infection. Journal of Inflammation (London, England), 20, 30.
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5

Mitochondrial Dynamics and Autophagy Regulation

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The antibodies used in our experiments were: anti-FUNDC1 antibody (Sigma, ABC506), anti-LC3 antibody (Proteintech, 14600-1-AP), anti-p62 antibody (Proteintech, 18420-1-AP), anti-LAMP1 (Boster, M00780-1), anti-LAMP2 (Proteintech, 27823-1-AP), anti-PDK4 antibody (Boster, BA3144), anti-cathepsin-D antibody (Boster, PB0020), anti-TFEB antibody (Proteintech, 13372-1-AP), anti-UQCRC1 antibody (Proteintech, 21705-1-AP), anti-Nrf2 antibody (Proteintech, 16396-1-AP), anti-Keap1 antibody (Proteintech, 10503-2-AP), anti-IP3R1 antibody (Boster, PB0223), anti-VDAC1 antibody (Proteintech, 10866-1-AP), anti-Mfn1 antibody (Proteintech, 13798-1-AP), anti-Mfn2 antibody (Proteintech, 12186-1-AP), anti-ATP6V01 antibody (Proteintech, 13828-1-AP), anti-LDHB antibody (Proteintech, 66425-1-Ig). Secondary antibody for western blotting: HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, SA00001-1), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Proteintech, SA00001-2). Secondary antibody for immunofluorescence: CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (Proteintech, SA00013-1), CoraLite594-conjugated Goat Anti-Rabbit IgG (H + L) (Proteintech, SA00013-4).
Reagents: DCA (Selleckchem, S8615, USA), 3-MA (Selleckchem, S2767, USA), Baf A1 (Selleckchem, S1413, USA), CQ (Selleckchem, S4430, USA), Lactate (Solarbio, L8630, China), 2-DG (GlpBio, GC17430, USA).
Ma W.Q., Sun X.J., Zhu Y, & Liu N.F. (2020). PDK4 promotes vascular calcification by interfering with autophagic activity and metabolic reprogramming. Cell Death & Disease, 11(11), 991.
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