Cells were lysed with RIPA buffer (Sangon), and proteins were quantified using a BSA Kit (Beyotime, Shanghai, China). Tumor proteins were extracted by a One-Step Animal Tissue Active Protein Extraction kit (Sangon). Protein (20 μg) was loaded and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Fdbio Science, Hangzhou, China) at 80 V for 30 min and then 120 V for 60 min followed by transfer to polyvinylidene fluoride (PVDF) membranes (Millipore) at 300 mA for 90 min. After blocking with 5% fat-free milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-Siglec-15 (1 : 1000, Santa Cruz), anti-GAPDH (1 : 5000, Sino Biological), anti-AGO2 (1 : 20, Abcam), anti-C-caspase3 (1 : 1000, Abcam), anti-caspase3 (1 : 1000, Abcam), and anti-Bcl-2 (1 : 1000, Abcam). After incubation with HRP-conjugated antibodies (1 : 5000, Sino Biological) for 2 h at room temperature, an ECL kit (Fdbio Science) was used for visualization of protein bands.
Sds page
Manufactured by Fdbio
Sourced in China
SDS–PAGE is a laboratory technique used to separate proteins based on their molecular weight. It utilizes an electric field to move charged molecules through a polyacrylamide gel matrix, allowing for the separation and analysis of complex protein mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Anti ago2, by Abcam (1 mentions)
Anti c caspase3, by Abcam (1 mentions)
Anti gapdh, by Sino Biological (1 mentions)
One step animal tissue active protein extraction kit, by Sangon (1 mentions)
Ecl kit, by Fdbio (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Ripa buffer, by Sangon (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
2 protocols using sds page
1
Protein Extraction and Western Blot Analysis
2
Analyzing Epithelial-Mesenchymal Transition in Liver Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The Huh7 and HepG2 liver cancer cells were harvested and lysed using radioimmunoprecipitation (RIPA; Beyotime, Shanghai, China) buffer containing 1% protease inhibitor phenylmethanesulfonylfluoride (PMSF; Beyotime). Following that, proteins were extracted, then quantified using the bicinchoninic acid (BCA; Beyotime) method. The same amount of protein was separated by 10% sodium-dodecyl-sulfate–polyacrylamide-gel electrophoresis (SDS-PAGE; Fdbio Science, Hangzhou, China) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Then, the membranes were incubated with TBST solution containing 5% FBS for 1 h to block the nonspecific antibody, followed by culturing with primary antibodies at 4°C overnight. The primary anti-human antibodies used (all from ABCAm; Cambridge, US) were E-cadherin (1: 1000), N-cadherin (1: 1000), Vimentin (1: 1000), Snail (1: 100), and GAPDH (1: 3000). Subsequently, we co-cultured the membranes with the secondary antibody (1: 10 000; Proteintech) for 1 h at room temperature. Finally, enhanced chemiluminescence (ECL; Millipore) was used to visualize the protein bands.
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