Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases
| Autor: | Angela Wandinger-Ness, Virginie Bondu, Tione Buranda, Peter C. Simons |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
RHOA Fluorophore biology medicine.diagnostic_test Chemistry Effector GTPase Primary and secondary antibodies Flow cytometry 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology 0302 clinical medicine 030220 oncology & carcinogenesis biology.protein Biophysics medicine Multiplex Rap1 |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493986118 |
| DOI: | 10.1007/978-1-4939-8612-5_13 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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