The protein sample was obtained from 0.1 g tissue samples with 1,000 μL RIPA lysis buffer containing 10 μL phenylmethanesulfonyl fluoride. The target proteins were isolated with SDS-PAGE and transferred onto PVDF membranes. Next, the membranes were sealed with 5% skim milk, and following primary antibodies were incubated overnight in a refrigerator (4℃): β-tubulin (1:1,800), β-actin (1:3,000), mTOR (1:600), ATG5 (1:1,000), and LC3B (1:1,000) from Abmart (Shanghai, China); Nrf2 (1:1,000) from Bioss (Beijing, China); Keap1 (1:1,800), GPx-1 (1:1,000), NQO1 (1:1,000), HO-1 (1:1,000), and p62 (1:800) from Wanleibio (Shenyang, China); and Beclin 1 (1:1,200) from ABclonal (Wuhan, China), all diluted in primary antibody dilution buffer (Beyotime, Shanghai, China). The membrane was incubated with the secondary goat anti-mouse antibody (1:5,000) and anti-rabbit IgG antibody (1:8,000), both from Bioss, for 1 h at 28℃. Lastly, the protein was identified using a hypersensitive chemiluminescence kit (Beyotime), and scanning and imaging was performed using a gel imaging system. Densitometric analysis of the protein bands was analyzed with ImageJ (Version 1.38) (Ali Shah et al., 2021 (link)).
Keap1
Manufactured by Wanlei
Sourced in China
Keap1 is a laboratory equipment product designed for use in scientific research. It serves as a key regulator within cellular signaling pathways. The core function of Keap1 is to modulate the activity of the Nrf2 transcription factor in response to cellular stress and redox changes.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using keap1
1
Quantification of Protein Expression Levels
2
Western Blot Analysis of Oxidative Stress Markers
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The total protein of each colon sample was extracted using RIPA lysis buffer, and the concentration and purity were measured using a BCA kit. SDS-PAGE and PVDF membrane were used to separate and transfer the proteins. The membranes were blocked with 5% skimmed milk powder for 2 h and incubated overnight at 4°C with the primary antibodies against Bax (1:1,000, Wanlei, China), Nrf2 (1:1,000, Wanlei), Gpx4 (1:5,000, Abcam, Britain), Keap 1(1:1,000, Wanlei), Ho-1 (1:2000, ABclonal, USA), and ß-actin (1:500, Wanlei). Subsequently, the membranes were washed three times with Tris-buffered saline Tween-20 (TBST) for 10 min and incubated with the secondary antibody (1:3,000, Zhongshan Jinqiao, China) at 25°C for 1 h. Membranes were washed three times with TBST, and protein signals were developed using an ECL kit. Images were captured using a gel imaging system and quantitatively analyzed using the ImageJ software. The experiment was performed in triplicate.
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