Hrp goat anti rabbit igg secondary antibody

The HeLa cells from the control and treated groups were harvested by scraping method. The harvested cells were added with 1X RIPA lysis buffer (ab156034, Abcam) and were incubated on ice for 30 minutes. The mixtures were centrifuged for 10 minutes at 12.000 rpm at 4°C. The supernatants were carefully collected to 1.5 mL tubes as total protein samples. The total protein quantification was performed using Pierce^TM BCA Protein Assay Kit (23227, Thermo Fisher Scientific). After being added with sample buffer, the samples were boiled for 10 minutes and centrifuged at 12.000 rpm for 10 minutes. The samples were loaded onto and were run by SDS-PAGE. The blots were transferred onto the nitrocellulose membrane. The blocking step was carried out using 5% of BSA for 1 hour. The membranes were washed out using PBS-Tween-20 (PBST), then incubated with primary antibodies specific against EGFR (A11352, Abclonal), ERK1/2 (A16686, Abclonal), CD133 (A0219, Abclonal), and β-actin (AC026, Abclonal) for overnight at 4°C. After the membranes were washed using PBST, the membranes were incubated with HRP Goat Anti-Rabbit IgG secondary antibody (AS014, Abclonal) for 1 hour at room temperature. To visualize the protein bands, the UVP Biospectrum Imaging System using SuperKinase^TM West Femto Maximum – ECL reagent (BMU102-EN, Abbkine).