Effects of Chemical Fixatives on Kinetic Measurements of Biomolecular Interaction on Cell Membrane

Abstract

Understanding the interaction between ligands and membrane proteins is important for drug design and optimization. Although investigation using live cells is desirable, it is not feasible in some circumstances and cell fixation is performed to reduce cell motion and degradation. This study compared the effects of five fixatives, i.e., formaldehyde vapor (FV), paraformaldehyde (PFA), acetone, methanol, and ethanol, on kinetic measurements via the LigandTracer method. We found that all five fixatives exerted insignificant effects on lectin-glycan interaction. However, antibody-receptor interaction is markedly perturbed by coagulant fixatives. The acetone fixation changed the binding of the anti-human epidermal growth factor receptor 2 (HER2) antibody to HER2 on the cell membrane from a 1:2 to a 1:1 binding model, while methanol and ethanol abolished the antibody binding possibly by removal of the HER2 receptors on the cell membrane. The capability of binding was retained when methanol fixation was performed at lower temperatures, albeit with a binding model of 1:1 instead. Moreover, whereas cell morphology does not exert a substantial impact on lectin-glycan interaction, it can indeed modify the binding model of antibody-receptor interaction. Our results provided insights into the selection of fixatives for cell-based kinetic studies.

Keywords: Cell fixation; Kinetics; Ligand binding; LigandTracer; Membrane protein.