Keap1-Nrf2 Association Analysis by Co-IP

As described [44 (link), 46 (link)], the Co-IP assay was performed to test the association between Keap1-Nrf2. In brief, OB-6 osteoblastic cells with applied treatment were lysed [44 (link)]. To the cleared lysates, 0.25 μg of Nrf2 antibody (Santa Cruz Biotech) was added per 0.8 mg of total cellular lysate proteins, and the immune complex formed by rotation for 24 hours at 4°C. The protein A/G-Sepharose (25 μL, Sigma) was then added and the incubation continued for additional 12 hours. The resulting immuno-precipitates captured with protein A/G-Sepharose were washed four times with CHAPS-containing buffer and analyzed by Western blotting assay.

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Guo S., Fei H.D., Ji F., Chen F.L., Xie Y, & Wang S.G. (2017). Activation of Nrf2 by MIND4-17 protects osteoblasts from hydrogen peroxide-induced oxidative stress. Oncotarget, 8(62), 105662-105672.

Publication 2017

Nrf2 antibody protein A/G-Sepharose

Antibody Assay Brief treatment Buffer Cellular Chaps Immune complex Keap1 Nrf2 Protein a sepharose Proteins Western blotting assay

Corresponding Organization :

Other organizations : Huaian First People’s Hospital, Nanjing Medical University

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Variable analysis

independent variables

Treatment applied to OB-6 osteoblastic cells

dependent variables

Association between Keap1-Nrf2

control variables

Cell type (OB-6 osteoblastic cells)
Lysis of cells
Addition of Nrf2 antibody (0.25 μg per 0.8 mg of total cellular lysate proteins)
Incubation of immune complex by rotation for 24 hours at 4°C
Addition of protein A/G-Sepharose (25 μL)
Incubation of protein A/G-Sepharose for additional 12 hours
Washing of immuno-precipitates captured with protein A/G-Sepharose four times with CHAPS-containing buffer
Analysis by Western blotting assay

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