E-cadherin Monoclonal Antibody (4A2C7)

Protocol tips

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	-Mouse (1:500)

Protocol tips
-Mouse (1:500)

Publication protocol

MCF-7 cells were plated in 24-well tissue culture plates. 24 hr later, they were serum starved with 0.1% FBS in DMEM with antibiotics. DECMA-1 (100 μg/mL), Sheep IgG control (100 μg/mL), GSI (1 uM), Z-DEV-FMK (50 μM) were also added at this time. DMSO was added as a control. Cells were lysed with RIPA containing protease inhibitors 24 hr after serum starvation and drug treatment. Cell lysates were run on a 4–20% SDS-PAGE gel with 2 mM sodium thioglycolate in the running buffer. The protein was then transferred to a nitrocellulose membrane using a Genie Blotter (Idea Scientific) and blocked with 5% milk in TBS-T. E-cadherin antibody was diluted 1:500, and the β-tubulin antibody was diluted 1:1000. A goat-anti mouse HRP conjugated antibody (Invitrogen) was used as a secondary antibody. Western blots were imaged using chemiluminescent buffer (Perkin Elmer Western Lightning Plus ECL) and the Amersham 600UV (GE) with staff support at the University of Minnesota-University Imaging Center.

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