AGS and HGC27 cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China). The membranes were blocked with 5% skimmed milk and incubated overnight at 4 ℃ with the following primary antibodies. The primary antibodies were as follows: anti-HMGB1 (1:1,000; Bioss, Beijing, China), anti-β-catenin (1:1,000; Bioss), anti-Wnt3a (1:1,000; Bioss), anti-c-Myc (1:1,000; Bioss), anti-E-cadherin (1:1,000; Servicebio), anti-N-cadherin (1:1,000; Servicebio), anti-Vimentin (1:1,000; Huabio, Hangzhou, China), anti-Snail (1:1,000; Huabio) and anti-GAPDH (1:4,000; GB11002, Servicebio) at 4 ℃ overnight. Then, the prepared membranes were incubated with secondary antibody (1:10,000, Bioss) for 2 h. Finally, the blots were visualized with an enhanced chemiluminescent reagent (Biosharp, Beijing, China).
Enhanced chemiluminescent reagent
Manufactured by Biosharp
Sourced in China
Enhanced chemiluminescent reagent is a laboratory product designed for the detection and quantification of target proteins or molecules in various biochemical and molecular biology applications. It is a sensitive and versatile solution that emits light upon reaction with the target analyte, allowing for the visualization and analysis of specific proteins or biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (1 mentions)
Anti gapdh, by Wuhan Servicebio Technology (1 mentions)
Anti β catenin, by Bioss Antibodies (1 mentions)
Anti e cadherin, by Wuhan Servicebio Technology (1 mentions)
Anti vimentin, by Huabio (1 mentions)
Anti n cadherin, by Wuhan Servicebio Technology (1 mentions)
Anti c myc, by Bioss Antibodies (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Bca protein assay reagent, by Beyotime (1 mentions)
Gapdh antibody, by Affinity Biosciences (1 mentions)
2 protocols using enhanced chemiluminescent reagent
1
Protein Expression Analysis in Cancer Cells
2
Quantitative Analysis of Inflammatory Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total proteins were extracted using a protein extraction kit (Beyotime Institute of Biotechnology Co., Shanghai, China) according to the manufacturer’s instructions. Protein content was quantified using the BCA protein assay reagent (Beyotime Institute of Biotechnology Co., Shanghai, China), and protein was diluted to the same concentration. Protein (25 μg per lane) was separated using sodium dodecyl sulfate-polyacrylamide gels electrophoresis, and was electro-transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were then incubated with either TNF-α and TNFR1 antibodies (Wanleibio Biological Technology Co., Ltd., Shenyang, China), p-IκB α and IκB α antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA), or NF-κB p65 and p-NF-κB p65 antibodies (Abcam, Cambridge, UK). A GAPDH antibody (Affinity Bioscience (China) Co., Hong Kong, China) was used as the internal control. Subsequently the PVDF membranes were incubated with an appropriate secondary antibody (Affinity Bioscience (China) Co., Hong Kong, China). Finally, protein bands were observed using an enhanced chemiluminescent reagent (Biosharp Life Sciences, Hefei, China). Protein grey intensity was quantified by the Fiji/ImageJ-win 64 program normalized GAPDH levels. Each western blot was performed a total of three times.
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