Total protein was extracted from cultured cells and analyzed as previously described 27 (link), 28 (link). A total of 50 μg of protein extracts were loaded to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, USA). After incubated with the primary antibodies overnight at 4 ℃ and then with HRP conjugated secondary antibodies (Promega, USA) for 1 h at room temperature, the membranes were subjected to the ECL chemiluminescence system (Pierce, USA). All of the experiments were repeated thrice. Primary antibodies used: CBX8 from Sigma Aldrich; Snail, Slug and Vimentin from BD Biosciences; E-cadherin and N-cadherin from Cell Signaling Technology; CBX6, Fibronectin and GAPDH from Santa Cruz.
Protocol detail
Protein Expression Analysis by Western Blot
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PVDF membranes
HRP conjugated secondary antibodies
Snail
Vimentin
E-cadherin
N-cadherin
Fibronectin
GAPDH
A protein
Antibodies
Chemiluminescence
Cultured cells
E cadherin
Fibronectin
Gapdh
Membranes
N cadherin
Promega
Protein
Pvdf
Sds page
Slug
Snail
Vimentin
Corresponding Organization : Sun Yat-sen University Cancer Center
Other organizations : Jiangxi Provincial Cancer Hospital
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Variable analysis
independent variables
- Protein extraction method
- Protein expression levels of CBX8, Snail, Slug, Vimentin, E-cadherin, N-cadherin, CBX6, Fibronectin, and GAPDH
- Amount of protein loaded (50 μg)
- Gel type (10% SDS-PAGE)
- Membrane type (PVDF)
- Primary antibodies used
- Secondary antibody (HRP conjugated)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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