The purpose of the experiments proposed in this grant is to generate recombinant antibody-like proteins (intrabodies) that can label specific proteins of all types, including cytoplasmic and nuclear proteins, and extracellular epitopes on transmembrane proteins. Recently we have shown the utility of the intrabody approach by generating probes that recognize the postsynaptic proteins Gephyrin and PSD95. These probes work efficiently and can be used to visualize endogenous proteins in living organisms. However, the design of these intrabodies places constraints on the targets against which they can be made. In particular, target proteins must be fixed to the cytoskeleton of the cell. Here we propose to develop a new strategy for generating intrabodies that does not place any requirements on the type of target protein. To establish the efficacy of this strategy we will generate intrabodies against the motor protein Kinesin 1, the transcription factor AP-1 and an extracellular epitope of the AMPA receptor GluA1.
|Fan, Linlin Z; Nehme, Ralda; Adam, Yoav et al. (2018) All-optical synaptic electrophysiology probes mechanism of ketamine-induced disinhibition. Nat Methods 15:823-831|