Transfected HCT116‐CSCs were harvested and lysed with RIPA extraction reagent (Solarbio) supplemented with a protease inhibitor cocktail (Solarbio) and phenylmethylsulfonyl fluoride (Solarbio). Equal amount of total protein (30 μg) was separated by 10% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to the PVDF membranes with 0.22 μm in pore size (Millipore) and then incubated with specific antibodies against E‐cadherin (1:1000; GeneTex), N‐cadherin (1:1000; GeneTex) and vimentin (1:1000; GeneTex) at 4°C overnight, while the anti‐GAPDH antibody (1:5,000; CMCTAG, Inc.) served as a loading control. The immunoblots were then incubated with secondary antibodies. ECL chemiluminescence substrate (Millipore) was used for quantification by densitometry with the Quantity One software (Bio‐Rad Laboratories, Inc.).
Ecl chemiluminescence substrate
Manufactured by Merck Group
Sourced in United States
The ECL chemiluminescence substrate is a laboratory reagent used to detect and quantify proteins in Western blotting applications. It generates a luminescent signal in the presence of the target protein, which can be measured using a compatible detection system.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Solarbio (2 mentions)
Ripa extraction reagent, by Solarbio (2 mentions)
Vimentin, by GeneTex (2 mentions)
E cadherin, by GeneTex (2 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
N cadherin, by GeneTex (1 mentions)
2 protocols using ecl chemiluminescence substrate
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of EMT Markers
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Transfected cells were lysed with RIPA extraction reagent (Solarbio; Beijing, China) supplemented with a protease inhibitor cocktail (Solarbio; Beijing, China) and phenylmethylsulfonyl fluoride (Solarbio, Beijing, China). Total protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the immunoblots were transferred to the PVDF membranes with 0.22 μm in pore size (Millipore; Billerica, MA, USA). Then, the immunoblots were incubated with specific antibodies against E-cadherin (GeneTex; Irvine, CA, USA) and Vimentin (GeneTex; Irvine, CA, USA), while GAPDH (CMCTAG, Inc.; Shanghai, China) served as a loading control. ECL chemiluminescence substrate (Millipore; Billerica, MA, USA) was used to quantify the protein expression by densitometry with the Quantity One software (Bio-Rad Laboratories, Inc.; Hercules, CA, USA).
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