In-Cell Western™ Assay Primary Antibody Selection and Validation

This application note describes important considerations for selecting and validating a primary antibody for the In-Cell Western™ (ICW) Assay. Antibody selection and validation are critical for development of a high-quality In-Cell Western assay. One major difference between conventional Western blots and the In-Cell Western is the context in which proteins are analyzed. ICW assays are performed on cultured cells that have been fixed to the bottom of a microplate. Fixed antigens have a slightly different conformational structure than those that have been removed from the cell and processed by SDS-PAGE. Consequently, some primary antibodies that perform well for Western blotting may exhibit poor binding characteristics on fixed protein epitopes, resulting in low or absent fluorescent signal during the detection step.