RIPA lysis buffer (Beyotime Biotechnology, China) containing 1% Halt™ Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific) was used to lyse cells. Pierce BCA Protein assay kit (Thermo Fisher Scientific, USA) was used to measure the protein concentration. The protein (40 μg) was separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The membrane was blocked in TBST containing 3% BSA and primary antibodies were incubated overnight at 4 °C. The primary antibodies were used as follows: anti-SIRT1, anti-p53, anti-Ac-p53, anti-p-p53, anti-p-Chk2, anti-p21, anti-TRAF2, anti-p-ATM, anti-Cyclin B1 (1:1000, Abcam) and anti-GAPDH (1:5000, Huabio). Subsequently, TBST was used to wash the membranes three times and the membranes were incubated at room temperature for 1 h with 1:3000 horseradish peroxidase conjugated anti-rabbit or mouse immunoglobulin G (Southern Biotechnology Associates, USA), followed by three washings in TBST. Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare) were used to visualize the specific bands with a ChemiScope 3300 Mini equipment (CLINX, China). The same membrane loaded with GAPDH served as the control.
Chemiscope 3300 mini equipment
Manufactured by Clinx
Sourced in China
The ChemiScope 3300 Mini is a compact and versatile laboratory equipment designed for general analytical applications. It features a high-resolution camera, advanced optics, and user-friendly software for capturing and analyzing images. The core function of the ChemiScope 3300 Mini is to provide efficient and reliable image acquisition and data processing capabilities for researchers and laboratory professionals.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Solarbio (3 mentions)
Ecl reagent, by Absin (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Huabio (1 mentions)
Anti p21, by Abcam (1 mentions)
Anti traf2, by Abcam (1 mentions)
Anti sirt1, by Abcam (1 mentions)
Anti p p53, by Abcam (1 mentions)
Anti p atm, by Abcam (1 mentions)
Anti cyclin b1, by Abcam (1 mentions)
Amersham ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
Anti ac p53, by Abcam (1 mentions)
Anti p53, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor cocktail 100, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab124964, by Abcam (1 mentions)
Ab215715, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab260043, by Abcam (1 mentions)
5 protocols using chemiscope 3300 mini equipment
1
Western Blot Analysis of Cellular Signaling
2
Comprehensive Protein Expression Analysis
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Total protein from cells was extracted with RIPA buffer (abs9229, Absin, China). The extracted protein was centrifuged and then quantified with the BCA kit (pc0020, Solarbio, China). After electrophoresis, they were electrotransferred onto the nitrocellulose membrane. Then, 5% nonfat milk was taken to block the membrane. After rinsing, the blocked membrane was subjected to primary antibodies at 4°C all night. The next day, an antirabbit secondary antibody (ab7090, Abcam, UK) was added. After reacting at 37°C for 1 h, a color reagent (1705061, BIO-RAD, USA) was taken to visualize the blots. Finally, the blots were developed in ChemiScope 3300 mini equipment (Clinx, China). The primary antibodies of collagen II (1: 1000, ab34712), fibronectin (1: 1000, ab268020), α-SMA (1: 50000, ab124964), Notch1 (1: 2000, ab52627), Jag1 (1: 500, ab7771), HEY1 (1: 3000, ab154077), HES1 (1: 1000, ab108937), TGF-β (1: 1000, ab215715), E-cadherin (1: 50000, ab40772), vimentin (1: 5000, ab92547), N-cadherin (1: 20000, ab76011), collagen I (1: 1000, ab260043), and GAPDH (ab181602) were bought from Abcam (UK).
3
Western Blot Protein Analysis
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Proteins extracted from cells and tissues were boiled for 10 min at 100 ℃ and resolved on 4‐20% gradient SDS‐PAGE gel (Genescript) subsequently transferred to PVDF membranes. The membranes were blocked in 1XTBS‐T containing 5%BSA for 40 minutes and incubated in primary following corresponding secondary antibodies. The bands were visualized by ChemiScope 3300 Mini equipment (CLINX, Shanghai, China) using EZ‐ECL Kit HRP (Biological industries, Israel).
4
Protein Expression Analysis of Ileal Tissues
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The harvested terminal ileum tissues were lysed via RIPA buffer (R0010, Solarbio, China). Next, we utilized the BCA kit (pc0020, Solarbio, China) to measure the concentrations of protein. The proteins were subjected to protein electrophoresis. Thereafter, the protein in the gel was shifted to a nitrocellulose membrane. After that, we utilized 5% nonfat milk to seal the membrane. The sealed membrane was washed and then exposed to primary antibodies all night at 4°C. The next day, the bound antibodies reacted with the anti-rabbit secondary antibody for 1.5 h at 37°C. After visualizing with the ECL reagent (abs920, Absin, China), the blots were placed in a ChemiScope 3300 Mini equipment (Clinx, China). The primary antibodies of anti-NLRP3 antibody (DF7438), anti-TLR4 antibody (AF7017), anti-NF-κB antibody (AF5006), anti-caspase-1 antibody (AF5418), and anti-GAPDH antibody (AF7021) were acquired from Affinity (USA).
5
Western Blot Analysis of EMT and Apoptosis Markers
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After treatment, cells and tissues were treated with RIPA buffer (P0013D, Beyotime, China) containing PMSF (ST506, Beyotime, China) to acquire total proteins followed by quantification with BCA kit (pc0020, Solarbio, China). After denaturation and electrophoresis, the protein was transferred to a nitrocellulose membrane. After blocking, the nitrocellulose membranes were exposed to specific primary antibodies overnight at 4°C. The next day, the bound antibodies were subjected to an anti-rabbit secondary antibody for 90 min at 37°C. In the end, signals were examined by an ECL reagent (abs920, absin, China) with ChemiScope 3300 mini equipment (Clinx, China). The primary antibodies of anti-E-cadherin antibody (1:40000), anti-Vimentin antibody (1:5000), anti-Fibronectin antibody (1:1000), anti-Snail antibody (1:1000, ab216347), anti-AKT antibody (1:500, ab8805), anti-p-AKT antibody (1:1000, ab38449), anti-GSK3β antibody (1:8000, ab32391), anti-p-GSK3β antibody (1:10000, ab75814), anti-Bax antibody (1:2000, ab182733), anti-Bcl-2 antibody (1:1000, ab32124), anti-β-actin antibody (1:200, ab115777) were obtained from Abcam (United Kingdom).
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