Western Blotting Protocol v1

This protocol describes culturing adherent cells, harvesting whole-cell protein lysates by RIPA lysis after drug or transfection treatments, quantifying protein by BCA, and performing SDS-PAGE and membrane transfer for Western blot detection. Membranes are blocked and probed with primary and secondary antibodies, followed by chemiluminescent (or fluorescent) visualization and optional re-probing. Expected results are consistent, comparable protein inputs across conditions (normalized to concentration) and clear, well-resolved target bands at the expected molecular weight with minimal background. Treatment-dependent changes (e.g., phosphorylation shifts after short serum-starve/drug exposure or expression changes after longer treatments) should be detectable as reproducible differences in band intensity or mobility between conditions.