3.4 Assay Runtime
Peter Simons, Virginie Bondu, Angela Wandinger-Ness, Tione Buranda
Rho GTPase
Rab GTPase
Cell signaling
Cytoskeleton
Hantavirus
Flow cytometry
Integrin activation
Sepsis
Multiplex
Protease-activated receptors
PARs
Thrombin
Argatroban
Bead functionalization
Glutathione-S-transferase
GST
GTPase effector beads
Rap1
RhoA
Rac1
Rab7
Fluorescence calibration beads
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.
Section 3.4 'Assay Runtime' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee
Steps
3.4 Assay Runtime
Just before the addition of lysates, mix the five different effector beads, centrifuge at 3000x g, and reduce the supernatant to about 5 μL. Resuspend the beads in 65µL, giving approximately 0.8nanomolar (nM). Add 5µL to twelve 0.65 mL microcentrifuge tubes for 12 multiplex assays. Leftover beads can be used later to set up the cytometer.
Add 50µL to each of the 12 tubes, mix, and rotate for 1h 0m 0s. When prepared as in Section 3.2 (Protocol "Production of a Cleared Cell Lysate"), there is enough of each lysate for triplicate determinations.
Centrifuge the tubes at 5000x g, reduce the supernatant to about 5 μL, and resuspend the beads in the residual volume.
Wash the beads with 50µL to block the beads’ nonspecific antibody-binding sites, centrifuge at 3000x g, and resuspend in 5µL ( see Note 5 ).
Add 50µL (1:20 dilution) against the 5 GTPases to each tube, mix, and rotate the tubes for 1h 0m 0s.
Wash the beads with 50µL, centrifuge at 3000x g, and resuspend in 5µL. Add 50µL (1:100 dilution) to each tube, mix, and rotate the tubes for 1h 0m 0s.
Centrifuge at 3000x g, reduce the supernatants to 5 μL, and resuspend the beads in 5µL.
Dilute the beads from each tube with 100µL just before each flow cytometric reading.
