Total egfr antibody

To prepare total protein lysates, colonoids were harvested using BD Cell Recovery Solution in order to achieve Matrigel matrix depolymerization. The wells were washed with cold PBS and incubated for 60 minutes in cold BD Cell Recovery Solution. Once the colonoids were released, the cells were centrifuged at 800rpm for 5 minutes at 4°C. The pellet was homogenized in lysis buffer containing a mixture of proteinase and phosphatase inhibitors and then centrifuged at 15,000rpm for 15 min at 4°C. The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL). Total protein was resolved on 4–12% precasted SDS-PAGE gels, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF membrane was blocked with 5% non-fat milk in PBS containing 0.1% Tween 20 for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies. The following antibodies were used in this study: GAPDH, anti-EGFR (phosphor Y1068) (1:2000, abcam, USA), total-EGFR, P-ERK, total-EGFR antibody (1:1000, cell signaling, USA). After washing with TBST, the blots were incubated for 2h at room temperature with HRP-conjugated secondary antibody (1:5000; Amersham Biosciences, San Francisco, CA, USA), visualized by using Electro-Chemi-Luminescence (ECL) chemiluminescent detection system (Amersham Biosciences).