‘Shotgun immunological’ approach for analysis of a complex subcellular system

Abstract

Abstract. Subsets of proteins present and the interactions between them are fundamental determinants of the properties of complex biological systems. Monoclonal antibodies (mAbs) are highly versatile tools for characterisation of such systems, being employed to analyse the structures, functions, locations and macromolecular interactions of their cognate antigens. However, production of mAbs using hybridoma technology is time-consuming, technically demanding and uses a large amount of target material. The study presented here demonstrates that a panel of synthetic single-chain fragment variable (scFv) mAbs recognising protein components of isolated terminal cisternae sarcoplasmic reticulum membranes can be rapidly selected by bacteriophage display, using small quantities of target material. The panel of scFv mAbs isolated proved useful in a wide range of immunological applications, including immunoblot, indirect immunofluorescence microscopy and for immunoprecipitation combined with identification of targets by mass spectroscopy. Such 'shotgun immunological' strategies will prove effective in characterising novel constituents of, as well as for investigating proteinprotein interactions within, macromolecular structures isolated from biological systems.