abstract

• Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.

authors

• Bondu, Virginie
• Buranda, Tione
• Simons, Peter C
• Wandinger-Ness, Angela

publication date

• January 1, 2018

published in

• Methods in molecular biology (Clifton, N.J.)  Journal

keywords

• Animals
• Antibodies, Monoclonal
• Cell Line
• Flow Cytometry
• Fluorescent Dyes
• GTP Phosphohydrolases
• Glutathione Transferase
• Guanosine Triphosphate
• Humans

Digital Object Identifier (DOI)

• https://doi.org/10.1007/978-1-4939-8612-5_13

PubMed ID

• 30062413

start page

• 177

end page

• 195

volume

• 1821

number