TITLE: 3.4 Assay Runtime
AUTHORS: Peter Simons, Virginie Bondu, Angela Wandinger-Ness, Tione Buranda

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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-</span><span style = "font-style:italic;">S</span><span>-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.</span></div><br/><br/><div class = "text-block"><span>Section 3.4 'Assay Runtime' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' </span><a href="https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee</span></a></div></div>

[STEPS]
?. [3.4 Assay Runtime]
Just before the addition of lysates, mix the five different effector beads, centrifuge at , and reduce the supernatant to about 5 μL. Resuspend the beads in , giving approximately . Add  to twelve 0.65 mL microcentrifuge tubes for 12 multiplex assays. Leftover beads can be used later to set up the cytometer.
Centrifuge: 3000 34, 2 min
[HPSMT]
[each GST-effector]
[suspension]
?. [3.4 Assay Runtime]
Centrifuge the tubes at , reduce the supernatant to about 5 μL, and resuspend the beads in the residual volume.
Centrifuge: 5000 34, 2 min
?. [3.4 Assay Runtime]
Add  to each of the 12 tubes, mix, and rotate for . When prepared as in Section 3.2 (Protocol "Production of a Cleared Cell Lysate"), there is enough of each lysate for triplicate determinations.
[cleared lysate]
?. [3.4 Assay Runtime]
Wash the beads with  to block the beads’ nonspecific antibody-binding sites, centrifuge at , and resuspend in  (see Note 5).
[blocking buffer #2]
Centrifuge: 3000 34, 2 min
[ residual buffer]
?. [3.4 Assay Runtime]
Add  (1:20 dilution) against the 5 GTPases to each tube, mix, and rotate the tubes for .
[primary antibodies]
?. [3.4 Assay Runtime]
Wash the beads with , centrifuge at , and resuspend in . Add  (1:100 dilution) to each tube, mix, and rotate the tubes for .
[blocking buffer #2]
Centrifuge: 3000 34, 2 min
[ residual buffer]
[goat anti-mouse antibody fluorescently labeled with Alexa488]
?. [3.4 Assay Runtime]
Centrifuge at , reduce the supernatants to 5 μL, and resuspend the beads in .
Centrifuge: 3000 34, 2 min
[ residual buffer]
?. [3.4 Assay Runtime]
Dilute the beads from each tube with  just before each flow cytometric reading.
[ blocking buffer #1]