A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of developed target protein. Due its several advantages over monoclonal antibodies, development has gained enormous popularity among cancer researchers. Systematic evolution ligands by exponential enrichment (SELEX) most common method aptamers specific for proteins interest. Following SELEX, a quick efficient binding assay accelerates process identification, confirming aptamer. This paper explains step-by-step flow cytometric-based epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM overexpressed carcinomas plays roles initiation, progression, metastasis. Therefore, it valuable candidate targeted drug delivery tumors. To evaluate membrane-bound EpCAM, EpCAM-positive -negative cells are required. Additionally, non-binding with similar length 2-dimensional (2D) structure EpCAM-binding includes different buffers (blocking buffer, wash incubation FACS buffer) steps. incubated cell lines. washing steps, will be evaluated using sensitive cytometry assay. Analysis results shows EpCAM-specific not EpCAM-negative cells. In cells, this depicted as band shift right compared control. corresponding bands -non-binding overlap. demonstrates While protocol focused on aptamer, applicable other published aptamers.