Total protein was extracted from cells using pre-cooled RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). Protein quantification was conducted with the Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific). An equal amount of protein samples was separated by 4-12% SDS-PAGE (GenScript, Nanjing, China) and then transferred to 0.45-μm PVDF membranes (Millipore, Billerica, MA, USA). After being blocked using 5% (v/v) non-fat milk in TBST for 1 h, membranes were incubated with corresponding primary antibodies at 4 °C overnight. Next, they were washed with TBST thrice and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoblots were detected with an imaging system (Bio-Rad, Hercules, CA, USA) using an enhanced chemiluminescence detection kit (Servicebio, Wuhan, China). GAPDH and α-tublin were selected to be the loading controls.
Enhanced chemiluminescence detection kit
Manufactured by Wuhan Servicebio Technology
Sourced in United States
The Enhanced Chemiluminescence Detection Kit is a laboratory equipment product used for the detection and quantification of proteins in western blot analysis. It utilizes a chemiluminescent substrate to generate a light signal proportional to the amount of target protein present in the sample, enabling sensitive and accurate protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Sds page, by GenScript (4 mentions)
0.45 μm pvdf membrane, by Merck Group (4 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti phospho pi3k tyr485 sc 130211, by Santa Cruz Biotechnology (1 mentions)
Anti gadph, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
4 protocols using enhanced chemiluminescence detection kit
1
Protein Extraction and Western Blotting
2
Western Blot Analysis of Cellular Proteins
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First, we used precooled RIPA buffer containing protease inhibitor (Thermo Scientific, USA) (Beyotime, Shanghai, China) to extract total protein from cells. Equivalent amounts of protein samples were isolated with 4-12% SDS-Page (GenScript, Nanjing, China) and then transferred to 0.45μm PVDF membrane (Millipore, USA). The membrane was sealed with TBST containing 5% skim milk for two h and incubated with primary antibody at four °C overnight. After washing with TBST 3 times, the antibody was coupled with HRP and incubated for one h at room temperature. Immunoblots were detected by an imaging system (Bio-Rad, USA) using an enhanced chemiluminescence detection kit (Servicebio, Wuhan, China). Western blots were performed using an imaging system (Bio-RAD, USA) using an enhanced chemiluminescence assay kit (Servicebio, Wuhan, China). The primary antibodies consisted of beta-catenin (Proteintech, 51067-2-AP), cyclin D1 (Cell Signaling Technology, 55506S), and GAPDH (Cell Signaling Technology, 5174S). The above antibodies are used in accordance with manufacturer’s agreement and instructions
3
Quantitative Protein Analysis by Western Blotting
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Total protein was extracted from tissues or cells using pre-cooled RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Thermo Scientific, USA). Quantification of protein was conducted with Bicinchoninic Acid Protein Assay Kit (Thermo Scientific, USA). An equal amount of protein samples was separated by 4–12% SDS-PAGE (GenScript, Nanjing, China) and then transferred to 0.45 μm PVDF membranes (Millipore, USA). After being blocked by 5% non-fat milk in TBST for 1 h, membranes were incubated with corresponding primary antibodies at 4 °C overnight. Washed by TBST for three times, they would be incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoblots were detected with an imaging system (Bio-Rad, USA) using enhanced chemiluminescence detection kit (Servicebio, Wuhan, China). GAPDH and β-actin were selected to be the loading controls. All the antibodies employed in this study were listed in Additional file 2 : Table S2.
4
Western Blot Analysis of Protein Markers
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Total proteins were extracted from tissues or cells using pre-cooled RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors (Thermo Scientific, USA). Protein quantification was performed with a dicinchoninic acid protein assay kit (Thermo Scientific, USA). Equal amounts of protein samples were separated by the 4-12% SDS-PAGE (GenScript, Nanjing, China) and then transferred to 0.45μm PVDF membranes (Millipore, USA). After blocking with TBST containing 5% skim milk for 2h, the membranes were incubated with the corresponding primary antibodies overnight at 4°C. TBST was washed 3 times and incubated with HRP-coupled secondary antibodies at room temperature for 1h. Immunoblots were detected by an imaging system (Bio-Rad, USA) using an enhanced chemiluminescence detection kit (Servicebio, Wuhan, China). GAPDH was selected as a loading control. Primary antibodies specific for CHPF (ab224495) were purchased from Abcam. Anti-GADPH (#51332), anti-E-cadherin (#2195), anti-N-cadherin (#13116), anti-Vimentin (#5741), anti-Snail (#3879), anti-PI3K(#4249), anti-AKT(#4691), and anti-p-AKT(#S473) were purchased from Cell Signaling Technology. Anti-phospho-PI3K(Tyr485) (sc-130211) was purchased from Santa Cruz Biotechnology (USA). The secondary goat anti-rabbit or goat anti-mouse (Abcam, USA).
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