Nrf2 primary antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The Nrf2 primary antibody is a research-use only tool designed to detect the Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2) protein in various biological samples. Nrf2 is a transcription factor that plays a crucial role in the regulation of antioxidant response elements and the expression of genes involved in cellular defense against oxidative stress.

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Lab products found in correlation

Lsm 710 nlo confocal microscope, by Zeiss (1 mentions) Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions) Alexa fluor 594 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions) Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions) Nis elements ar software, by Nikon (1 mentions) Evans blue, by Merck Group (1 mentions) Streptozotocin (stz), by Merck Group (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Cell counting kit 8 cck 8, by Beyotime (1 mentions) Gapdh primary antibody, by Proteintech (1 mentions) Supersignal west femto maximum sensitivity substrate kit, by Thermo Fisher Scientific (1 mentions) Immunobilon p membrane, by Merck Group (1 mentions) Mouse antibody against β actin, by Abcam (1 mentions)

5 protocols using nrf2 primary antibody

Nrf2 Nuclear Localization Assay with Sch A Pretreatment

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Nuclear localization of Nrf2 was detected by immunofluorescence confocal microscopy. Cells were exposed to DON at 1 μM for 3 hours for Nrf2. The duration of DON treatment for Nrf2 was determined based on our preliminary experiments (data not shown). For the Sch A pre-treated samples, 10 μM Sch A exposure was carried out for 24 hours, followed by DON exposure at 1 μM for 3 hours. Cells were also exposed to 10 μM Sch A alone for 24 hours. After treatment, cells were fixed with 10% (v/v) formalin and ice cold absolute methanol, followed by permeabilization with 0.1% Triton X-100. They were then incubated with Nrf2 primary antibody (1:500 dilution) (#12721; Cell Signalling) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077, Abcam) as secondary antibody (1:200), and subsequently Hoechst staining and viewed and analyzed in a Zeiss LSM 710 NLO confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Wan M.L., Turner P.C., Co V.A., Wang M.F., Amiri K.M, & El-Nezami H. (2019). Schisandrin A protects intestinal epithelial cells from deoxynivalenol-induced cytotoxicity, oxidative damage and inflammation. Scientific Reports, 9, 19173.

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Nrf2 Immunofluorescence Staining Protocol

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For Nrf2 staining, 2000 cells of BEAS-2B and Cr-T were seeded on 8-well
chambered coverglass slides (ThermoScientific) and incubated overnight at
37°C. Then, cells were fixed with 4% paraformaldehyde PBS (pH 7.4) for 10
minutes and permeabilized with 0.1% Triton X-100 for 20 minutes at RT. Cells
were incubated at 4°C overnight with Nrf2 primary antibody (#12721, Cell
Signaling Technology) after blocking with 1% BSA, 22.52 mg/ml glycine in PBST
(PBS+0.1% Tween 20). Following day, cells were washed with PBS three times and
labeled with Alexa Fluor 594-conjugated goat anti-mouse IgG secondary antibody
(A-11005, Invitrogen) at RT for 1 hour. ProLong™ Diamond Antifade
Mountant with DAPI (ThermoScientific Rockford, IL) was used for staining cell
nuclei and mounting the samples. Fluorescence images were acquired using a Nikon
DS-Ri2 and analyzed in Nikon NIS-Elements AR software.

Wang L., Bayanbold K., Zhao L., Wang Y., Adamcakova-Dodd A., Thorne P.S., Yang H., Jiang B.H, & Liu L.Z. (2021). Redox sensitive miR-27a/b/Nrf2 signaling in Cr(VI)-induced carcinogenesis. The Science of the total environment, 809, 151118.

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Nrf2 Expression in Rat Liver

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The liver tissues of rat were fixed in 4% paraformaldehyde overnight, followed by embedment in paraffin and longitudinal slicing, with 4 μm thick sections obtained and stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) staining was conducted to examine the expression of Nrf2 in the liver. Briefly, the sections were deparaffinized and rehydrated, then incubated with a hydrogen peroxide block for 15 min. The sections were incubated with Nrf2 primary antibody (1:500, Cell Signaling Technology) overnight at 4°C followed by incubation in biotinylated secondary antibody for 1 hr at room temperature. After incubation with DAB chromogen, the sections were ultimately counterstained with hematoxylin. The stained slides were examined for histomorphological analyses at 400× magnification using optical microscope.

Xiong Y., Wang Y., Xiong Y, & Teng L. (2021). Protective effect of Salidroside on hypoxia‐related liver oxidative stress and inflammation via Nrf2 and JAK2/STAT3 signaling pathways. Food Science & Nutrition, 9(9), 5060-5069.

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Streptozotocin-Induced Ferroptosis Modulation

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Streptozotocin (STZ), triphenyl tetrazolium chloride (TTC), and Evans blue (EB) were purchased from Sigma Chemical Co. (MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Laboratories (Grand Island, NY, USA). Sulforaphane (SFN), the NRF2 activator, was purchased from Glpbio Co. (CA, USA). Erastin (Era), the ferroptosis inducer, was purchased from Selleck (Houston, TX). The cell counting kit-8 (CCK-8) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay test kits were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), superoxide dismutase (SOD), and malondialdehyde (MDA) assay test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Fe²⁺ assay test kit was purchased from Abcam Co. (Abcam, UK). The primers for NRF2, FPN1, ACSL4, and β-actin were designed and synthesized by Wuhan Servicebio Co. (Wuhan, Hubei, China). NRF2 primary antibodies were purchased from Cell Signaling Technology (CST, Beverly, CA, USA). FPN1, ACSL4, GPX4, and GAPDH primary antibodies were obtained from Proteintech Co. (Wuhan, Hubei, China).

Tian H., Xiong Y., Zhang Y., Leng Y., Tao J., Li L., Qiu Z, & Xia Z. (2022). Activation of NRF2/FPN1 pathway attenuates myocardial ischemia–reperfusion injury in diabetic rats by regulating iron homeostasis and ferroptosis. Cell Stress & Chaperones, 27(2), 149-164.

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Nrf2 Protein Expression Analysis

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Equal amounts of protein (30 or 50 μg) from cultured cells and embryos were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immunobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated in 5% nonfat milk for 1 hour, and then incubated for 18 hours at 4°C with Nrf2 primary antibodies (Cell Signaling Technology, Danvers, MA) at dilutions of 1:1000 in 5% nonfat milk. Membranes were then exposed to goat anti-rabbit secondary antibodies. To ensure that equivalent amounts of protein were loaded, membranes were stripped and probed with a mouse antibody against β-actin (1:5000; Abcam, Cambridge, UK). Signals were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Scientific, Waltham, MA). Quantification of blots was performed using VisionWorksLS software (UVP Company, Upland, CA). All experiments were repeated in triplicate.

Zhao Y., Dong D., Reece E.A., Wang A.R, & Yang P. (2017). Oxidative stress-induced miR-27a targets the redox gene Nrf2 in diabetic embryopathy. American journal of obstetrics and gynecology, 218(1), 136.e1-136.e10.

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