Generation of functional antibodies for mammalian membrane protein crystallography

•Antibody fragments facilitate crystallization of membrane proteins.•We present our protocols to generate functional antibodies against membrane proteins.•Crystal structure of a human GPCR in complex with Fab fragment has been obtained.•Tight binding of the functional antibody locks the GPCR into an inactive state.

Membrane proteins act as gateways to cells, and they are responsible for much of the communication between cells and their environments. Crystallography of membrane proteins is often limited by the difficulty of crystallization in detergent micelles. Co-crystallization with antibody fragments has been reported as a method to facilitate the crystallization of membrane proteins; however, it is widely known that the generation of mouse monoclonal antibodies that recognize the conformational epitopes of mammalian integral membrane proteins is typically difficult. Here, we present our protocols to generate functional mouse antibodies for the membrane protein crystallography, which have enabled us to solve crystal structures of mammalian receptors and transporters complexed with antibody fragments.

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