3.3 Molecular Assembly of GST Effector Proteins on Glutathione Beads
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 3.3 'Molecular Assembly of GST Effector Proteins on Glutathione Beads' 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
3.3 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 4°C, collected by centrifugation, and resuspended in HPSMT buffer to 10,000 beads/μL. Beads are prepared fresh for each experiment and kept On ice 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 ( K d ∼ 80 nM) [26] measured for GST-GFP is used to estimate the occupancy of GSH sites on beads according to Eq. (1):
Site occupancies of ligand-binding sites at saturation are in the range of 1–4 × 106ligand 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 × 1010sites or 3.3 nM in 20 μL. Incubating 800 nM (10× K d) GST ligand with 10,000 beads is expected to yield an occupancy, θ , of 0.91 (or 91%).
12–18 h before a putative assay of 12 samples for 5 GTPase targets perform the following:
Mix 12µL (12 × 104beads) of each set of 700 nm color-coded glutathione beads with a tenfold excess 120µL for 0h 20m 0s at Room temperature, to block the nonspecific binding sites on the particles.
Centrifuge the beads at 5000x g, our standard.
Resuspend each of the five sets in 15µL. All further operations are performed at °C – 4°C.
Add five distinct GST-effector proteins (800nanomolar (nM)) 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 5000x g to reduce the supernatants to 5 μL.
Wash the bead sets. For this, add 50µL to each set and mix, centrifuge at 3000x g to reduce the supernatants to 5 μL, resuspend the pellet, and dilute it in 50µL, giving about 8nanomolar (nM). The beads are ready for use and can be stored at 4°C .
