Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types.

Abstract

The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous protein in living cells without overexpression of fusion proteins or genetic tagging has not been routinely possible. Here we describe a versatile antibody-based imaging approach (VANIMA) to be able to precisely locate and track endogenous proteins in living cells. The labeling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies or antibody fragments (Fabs) into living cells and the specific binding of these antibodies to the target protein inside of the cell. Our protocol describes step by step the procedure from testing of the suitability of the desired antibody, over the digestion of the antibody to Fabs until the labeling and the delivery by electroporation of the antibody or Fab into the cells. VANIMA can be adapted to any monoclonal antibody, self-produced or commercial, and many different metazoan cell lines. Additionally, our method is simple to implement and can be used not only to visualize and track endogenous factors, but also to specifically label posttranslational modifications, which cannot be achieved by any other labeling technique so far.