Protein was extracted from tissues using RIPA buffer. After denaturing at 95°C for 10 minutes, protein was separated on a 12% SDS‐polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. After blocking with TBST buffer containing 5% non‐fat milk for 1 hour at room temperature, the membrane was co‐incubated with primary antibodies: rabbit anti‐CAMK2N1 (PA5‐23740, 1:1000; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti‐p‐ERK1/2 (ab223500, 1:400; Cambridge, MA, USA), rabbit anti‐ERK1/2 (ab17942, 1:1000; Abcam), rabbit anti‐p‐EMK (ab60002, 1:500; Abcam), rabbit anti‐EMK (ab60002, 1:2000; Abcam), rabbit anti‐Bax (ab32503, 1:1000; Abcam), rabbit anti‐Bcl2 (Bcl2, 1:2000; Abcam) and GAPDH (ab8245, 1:500; Abcam) at 4°C overnight. The membrane was co‐incubated with HRP‐conjugated goat anti‐rabbit IgG (ab6721, 1:2000; Abcam) at 37°C for 1 hour after washing with TBST. Finally, the results were visualized using Chemical Mp Imaging System (Bio‐Rad, Hercules, CA, USA) and analysed by Gel‐ProAnalyzer (UnitedBio, USA). Protein levels were normalized to GAPDH.
Chemical mp imaging system
Manufactured by Bio-Rad
Sourced in United States
The Chemical Mp Imaging System is a laboratory instrument designed for protein separation and analysis. It utilizes gel electrophoresis and imaging technology to visualize and quantify protein samples. The core function of this system is to provide accurate and reliable data for researchers studying protein structures and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Supersignal west femto agent, by Merck Group (2 mentions)
Protease inhibitor, by CoWin Biotech (2 mentions)
Ab17942, by Abcam (1 mentions)
Ab223500, by Abcam (1 mentions)
Ab60002, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Ab49849, by Abcam (1 mentions)
Hrp labelled goat anti mouse iggs, by Abcam (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Bca kit, by Beyotime (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Anti gapdh, by Bioss Antibodies (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Bioss Antibodies (1 mentions)
Anti zeb1, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using chemical mp imaging system
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of AKAP12 Expression
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Protein was extracted from tissues using RIPA buffer containing 1/10 Complete Mini protease inhibitor cocktail (Roche, Basel, Switzerland). The protein content was measured with a BCA Protein Assay kit (Beyotime, Shanghai, China). After denaturing at 95°C for 10 minutes, 50 μg of each protein sample was separated on a 12% SDS‐polyacrylamide gel and then transferred to a polyvinylidenefluoride (PVDF) membrane. After blocking with 5% degrease milk in TBST buffer, the membrane was incubated with mouse monoclonal antibody to AKAP12 (ab49849, 1:200; Abcam, Cambridge, MA, USA) and GAPDH (ab8245, 1:500, Abcam) at 4°C overnight. After washing 3 times with TBST buffer every 15 minutes, the membrane was further incubated with HRP‐labelled goat antimouse IgGs (1:200; Abcam) at 37°C for 1 hour. Finally, the results of Western blot were scanned with Chemical Mp Imaging System (Bio‐Rad, Hercules, CA, USA) and analysed by Gel‐ProAnalyzer (software version 4; UnitedBio, USA). GAPDH was used as a loading control for Western blots.
3
Western Blot Detection of Protein Expression
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We used Radioimmunoprecipitation assay (RIPA) buffer to the lyse transfected cells, obtained the supernatant, and added protease inhibitors (1%; ComWin Biotech, Beijing, China). The protein concentration was assessed using a bicinchoninic acid (BCA) Kit (Beyotime, Jiangsu, China). Equal amounts of protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% or 8%) and transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes (Roche). After blocking with 5% nonfat milk for 1 h, the PVDF membranes were incubated with primary antibodies overnight at 4 °C. Membranes were washed a minimum of three times in Tris-buffered saline-Tween 20 (TBST) for 10 min each time, followed by 1 h of incubation with goat anti-rabbit or goat anti-mouse secondary antibodies. Finally, the membrane was washed three times with TBST again. The immunoreactive protein bands were detected using SuperSignal West Femto Agent (Millipore, Billerica, MA, USA) and visualized using the Chemical Mp Imaging System (Bio-Rad). The antibodies used are listed in Table S4 .
4
Probing Epithelial-Mesenchymal Transition Markers
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RIPA buffer was added to cells and incubated with shaking on ice for more than half an hour. The lysate supernatant was collected, and a protease inhibitor (1%; ComWin Biotech, Beijing, China) was added. The supernatant was centrifuged, and the protein concentration was measured using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated by 10% SDS-PAGE and were then transferred to PVDF membranes (Millipore, USA). After blocking for 1 h with 5% skim milk powder, membranes were incubated with the primary antibody at 4 °C overnight. The following primary antibodies were used: anti-ZEB1 (1:1000 dilution,; Proteintech, USA), anti-E-cadherin (1:1000 dilution, Cell Signaling Technology (CST), USA), anti-N-cadherin (1:1000 dilution; CST, USA), anti-vimentin (1:1000 dilution; Bioss, China), and anti-GAPDH (1:5000 dilution; Bioss, China). Membranes were washed three times for 10 min each in TBST containing 0.1% Tween and were then incubated with goat anti-rabbit or goat anti-mouse secondary antibodies (1:8000 dilution, Bioss, China) for 1 h at room temperature. Membranes were washed three times with TBST containing 0.1% Tween. Protein bands were detected with SuperSignal West Femto Agent (Millipore) and visualized with the Chemical Mp Imaging System (Bio-Rad).
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