Publication protocol
Proteins isolated from tissues and cell samples were promptly homogenized in lysis buffer which contained 1 M Tris-HCl pH 7.5, 1% Triton X-100, 1% Nonidet P-40 (NP-40), 10% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 0.5 M EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF and then centrifuged for 30 min at 10,000 × g to collect the supernatant liquid. The supernatants were stored at −80°C until use. The protein concentrations were measured with a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Equal amount of protein from each sample was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). After being blocked in 5% non-fat milk in TBST [150 mM NaCl, 20 mM Tris (pH 7.4) and 0.05% Tween-20] for 2 h, the membranes were first incubated with primary antibodies overnight at 4°C. The primary antibodies used in this report included the followed: anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cat. no. sc-47724; 1:3,000), anti-DTX3L (cat. no. sc-100627; 1:500), E-cadherin (cat. no. sc-8426; 1:500), vimentin (cat. no. sc-6260; 1:1,000) (all from Santa Cruz Biotechnology, Inc.), anti-caspase-3 (cleaved) (cat. no. 9661; 1:1,000), and anti-PARP (cleaved) (cat. no. 5625; 1:1,000) (both from Cell Signaling Technology, Danvers, MA, USA). After washing for three times, 5 min each, the membranes were then incubated with horseradish peroxidase-conjugated human anti-mouse (cat. no. A11126) or anti-rabbit (cat. no. 71-2700) antibodies (1:1,000; Pierce, Rockford, IL, USA) as the secondary antibody at room temperature for 2 h. Finally, the membranes were detected with ECL detection systems. The experiments were implemented in three independent reactions.
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