E-cadherin Antibody (G-10): sc-8426

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) --Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) --Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

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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Manufacturer protocol

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