Page 65 - Live-cellanalysis handbook

Kinetic Assays for Quantifying Protein Dynamics

Complementary, live-cell solutions for studying protein dynamics

Proteins – the building blocks of the cell – are required for possible to quantitatively link the dynamics of a target protein
virtually every biological process, from cell function, to structure with cell morphology, proliferation and function. The protocol is
and regulation. The study of proteins – how they move, interact simple despite the richness of data and insight generated, involving
and communicate with each other – is thus essential for our a single antibody-labeling step followed by direct addition to cells,
understanding of health and disease. thereby removing the need for laborious labeling and blocking
procedures and wash steps that can result in unwanted cellular
This is particularly true of proteins on the surface of cells involved changes and misleading results.
in cell signaling and ligand binding, which requires the interaction
of both extracellular proteins, such as cytokines and hormones, IncuCyte® FabFluor antibody labeling reagents are highly specific
and membrane proteins. Such interactions are often the drivers and sensitive, amenable to both adherent and non-adherent cell
of disease processes or the basis of drug mechanism of action. models. The data below exemplifies how the assays can be used
Antigen-mediated antibody internalization, for example, plays to study long-term protein dynamics and their relationship to cell
an important role in several antibody-based therapeutics and in function, morphology, as well as cell-cell interactions.
differentiation-induced protein expression changes.

However, despite their importance, our ability to analyze these
processes and link their properties to cell function has been Live-cell imaging and analysis approaches to
hindered by the methodologies available to study them. Current
methodologies for analysis of membrane proteins and their studying protein dynamics
receptor function are limited to single time point analyses, not
taking into account the dynamic changes in protein expression Fluorescently labeled antibodies are widely used as markers for
due to protein turnover or in response to biochemical cues. While visualizing cell surface protein expression, receptor interactions
traditional protein assays have provided insights into localization, and antibody internalization. To build on existing methodologies
binding and protein-protein interactions; these single snapshots for studying protein expression, a novel strategy was developed
in time cannot follow the dynamics of proteins in living cells under based on fluorescently labeled antibody fragments (Fabs) that
physiological conditions and therefore are lacking in physiological target the Fc region of any mouse, human or rat antibody. Two
context. types of reagents have been developed, differing in the type of
fluorescent molecule conjugated to the Fab and thereby enabling
IncuCyte’s live-cell analysis system and antibody labeling reagents two different applications, antibody internalization and live-cell
enable a new assay approach that delivers spatial and temporal immunocytochemistry. These two reagents, when combined with
information at scale in living cells. The unique antibody reagents the IncuCyte® Live-Cell Analysis System, provide an integrated
can be mixed with any Fc-containing antibody, applied directly to approach to studying protein dynamics in real-time and under
cells and then monitored under physiological conditions for hours physiological conditions.
or days. The real-time image acquisition and analysis means it is

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