Dc protein assay reagent kit

Harvested ES cells and EBs were lysed with RIPA lysis buffer (50mM Tris pH8.0, 150mM NaCl, 01% SDS, 0.5% Sodium deoxycholate, 1% NP40, complete protease inhibitor (Roche, 11697498001), and PhoSTOP phosphatase inhibitor (Roche, 04906845001)). A DC™ Protein assay reagent kit (BioRad, 500–0111) was used for measuring protein concentration. Equal protein amounts were separated by SDS/PAGE gel and transferred on PVDF membrane (BioRad, 1620177). The membranes were blocked and incubated overnight at 4^oC with 1:50 anti-Ezrin (Abcam, ab4069), 1:1000 anti-alpha-Tubulin (Abcam, ab15246), 1:1000 anti-E-cadherin, 1:1000 anti- ß-catenin, and 1:1000 anti-LC3 (MBL, PD014). Antibody binding was detected by ECL. Protein quantification was analyzed with Image J.