Western Blot Analysis of Protein Expression
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Other organizations : Chonnam National University
Variable analysis
- Transfected cells or xenograft tumor tissue samples
- Expression of ADAM12, MMP2, MMP9, E-cadherin, Snail, vimentin, claudin-1, integrin α5, integrin β1, integrin β3, phosphorylated (p)-AKT (S473), p-phosphoinositide-dependent protein kinase 1 (PDK1) (S241), p-glycogen synthase kinase-3β (GSK-3β) (S9), total AKT, total PDK1, total GSK-3β, and Myc-tag
- Phosphate-buffered saline (PBS)
- RIPA buffer with Halt™ phosphatase and protease inhibitor cocktail
- Protein quantification using BCA protein assay
- Separation of equal amounts (20 µg/lane) of protein lysate by electrophoresis on 8-12% polyacrylamide gels
- Transfer of proteins onto Immobilon®-P transfer membranes
- Incubation of blot membranes with 5% BSA solution
- Immunoblotting with specific antibodies (1:1,000 dilution) overnight at 4°C
- Washing of blot membranes with Tris-buffered saline-0.1% Tween-20 (TBS-T)
- Incubation with horseradish peroxidase-conjugated secondary antibody (1:2,000 dilution) for 1 h at room temperature
- Blot development using Amersham ECL Prime Western Blotting Detection Reagent
- Visualization of specific bands using LAS-400 luminescent image analyzer
- Semi-quantification of specific bands using Multi-Gauge gel analysis software
- Not specified
- Not specified
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