TITLE: 3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads
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 3.3 'Molecular Assembly of GST Effector Proteins on Glutathione Beads' 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]
?. [3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads]
Briefly, glutathione beads are mixed with the desired, purified GST-effector protein of desired concentration (Eq. 1), incubated with rotation  at , collected by centrifugation, and resuspended in HPSMT buffer to 10,000 beads/μL. Beads are prepared fresh for each experiment and kept  until use on the same day. It is desirable to use the beads as a limiting reagent. In this way, uniform site occupancy (θ in Eq. 1) of the GSH sites is assured for each bead preparation. The equilibrium dissociation constant (Kd ∼ 80 nM) [26] measured for GST-GFP is used to estimate the occupancy of GSH sites on beads according to Eq. (1):θ=([GST effector]_{free}/Kd)/(1+([GST effector]_{free}/Kd))Site occupancies of ligand-binding sites at saturation are in the range of 1–4 × 106 ligand sites/bead. Equation (1) can be used to estimate ligand-occupied sites according to the example: 10,000 beads present an upper limit of 4 × 1010 sites or 3.3 nM in 20 μL. Incubating 800 nM (10×Kd) GST ligand with 10,000 beads is expected to yield an occupancy, θ, of 0.91 (or 91%).
4 °C
on ice
?. [3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads]
12–18 h before a putative assay of 12 samples for 5 GTPase targets perform the following:
?. [3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads]
Mix  (12 × 104 beads) of each set of 700 nm color-coded glutathione beads with a tenfold excess  for  at , to block the nonspecific binding sites on the particles.
[beads]
[HPSMT blocking buffer #1]
0 Room temperature
?. [3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads]
Centrifuge the beads at , our standard.
Centrifuge: 5000 34, 2 min
?. [3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads]
Resuspend each of the five sets in . All further operations are performed at  – .
[residual buffer]
0 °C
4 °C
?. [3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads]
Add five distinct GST-effector proteins () separately to the five particle sets, where specific effector proteins are associated with the 700 nm intensity register of the bead. Mix the suspensions gently with a nutator . Centrifuge at  to reduce the supernatants to 5 μL.
Centrifuge: 5000 34, 2 min
?. [3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads]
Wash the bead sets. For this, add  to each set and mix, centrifuge at  to reduce the supernatants to 5 μL, resuspend the pellet, and dilute it in , giving about . The beads are ready for use and can be stored at .
[HPSMT]
Centrifuge: 3000 34, 2 min
[HPSMT]
[GST-effector]
4 °C