Cells were harvested and lysed with cell lysis reagent (NCM Biotech, Suzhou, China), and protein was collected. A total of 20 µg of total protein per lane was separated by 4-15% SDS-polyacrylamide gel electrophoresis and was then transferred to 0.2 μM or 0.45 μM PVDF membranes (Millipore, Billerica, USA). Subsequently, the membranes were blocked with 5% nonfat milk for 60-120 min. They were incubated separately at 4 °C overnight with the following primary antibodies: anti-BRD7 (Cat# 51009-2-AP, 1:500) and anti-GAPDH (Cat# 60004-1-Ig, 1:10,000) (Proteintech Group, Inc., Wuhan, China); anti-BIRC2 (Cat# 70008, 1:1,000), anti-E-cadherin (Cat# 14472, 1:1000) and anti-Snail (Cat# 3879, 1:1000) (purchased from CST, MA, USA); anti-Flag (Cat# F2555, 1:1000, Sigma-Aldrich, St. Louis, MO); anti-Vimentin (Cat# ARG66199, 1:1000, Arigo, Taiwan, China) and anti-N-cadherin (Cat# AF5239, 1:1000, Affinity Biosciences, Jiangsu, China). The membranes were then incubated with species-matched secondary antibodies for 60 min at 37 °C. Bands were visualized by Western Blotting Substrate (Share-Bio, Shanghai, China), and signals were detected by an enhanced chemiluminescence detection system according to the manufacturer’s protocol (MiniChemi™ I, SAGECREATION, China).
Minichemi 1
Manufactured by Sagecreation
Sourced in China
The MiniChemi™ I is a compact and versatile laboratory instrument designed for performing chemiluminescence-based assays. It is capable of detecting and quantifying light-emitting chemical reactions, making it a valuable tool for various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Anti gapdh, by Proteintech (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti e cadherin, by Merck Group (1 mentions)
Anti vimentin, by Arigo Biolaboratories (1 mentions)
Anti n cadherin, by Affinity Biosciences (1 mentions)
Anti snail, by Merck Group (1 mentions)
Anti brd7, by Proteintech (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti ccnb1, by Abways (1 mentions)
Anti chek1, by Abways (1 mentions)
Anti ccne1, by Abways (1 mentions)
Anti cdk1, by Abways (1 mentions)
Anti cdk4, by ABclonal (1 mentions)
Coomassie bradford protein assay kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using minichemi 1
1
Protein Expression Analysis by Western Blot
2
Protein Expression Analysis of Cell Lines
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Two cell lines (LM3 and HepG2) in the treatment groups were treated with the IC50 concentrations at 24 hours of two drugs (CI, Cino) for 24 hours and 48 hours, respectively, then the cell lines in the control groups and the treatment groups were digested and lysed respectively. Cell lysates were separated by electrophoresis on 10% SDS-polyacrylamide gels (NCM Biotech) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). The membranes were then blocked with 5% skim milk and incubated overnight at 4 °C with the following primary detection antibodies: anti-CCNB1 (dilution 1:1000; Abways, Shanghai, China), anti-CHEK1 (dilution 1:1000; Abways), anti-CCNE1 (dilution 1:400; Abways), anti-CDK1 (dilution 1:1000; Abways), anti-CDK4 (dilution 1:1000; ABclonal, Wuhan, China) and anti-GAPDH (dilution 1:5000; ProteinTech). Next, the membranes were further incubated with species-matched secondary antibodies for one and a half hours at 37 °C and then stained with western blotting substrate (Thermo Scientific, USA). Finally, band signals were detected with an enhanced chemiluminescence detection system according to the manufacturer’s protocol (MiniChemi™ I, Sage Creation, Beijing, China).
3
Western Blot Analysis of CADM1 Protein
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Cells were washed twice with PBS, and protein was harvested by using RIPA reagent containing protease inhibitor cocktail (ThermoFisher Scientific). Protein concentration was measured by using the Coomassie (Bradford) Protein Assay Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Equal quantities of protein were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA), which were blocked with 5% skim milk in TBST for 2 hrs at room temperature, then incubated with primary antibodies overnight at 4°C, followed by a TBST wash and secondary antibody incubation for 2 hrs at room temperature. Finally, membranes were incubated with Western LumaxLight Superior HRP substrate (ZETA LIFE Inc.) and protein bands were captured on a MiniChemi I instrument (SageCreation, Beijing). The primary antibodies used were anti-CADM1 (Proteintech, Cat#14335-1-AP) and anti-GAPDH (TransGen Biotec, Cat#HC301-02).
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