TITLE: 3.1 Synthesis of 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 3.1 'Synthesis of 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><br/></div>

[STEPS]
?. [3.1 Synthesis of Glutathione Beads]
High-site-density glutathione-derivatized beads used for flow cytometry have been synthesized previously from 13 μm dextran-cross-linked agarose beads [26, 27], and 4 μm amino polystyrene beads [20]. In this method, 5.4 μm Cyto-Plex™ carboxylated polystyrene bead sets are first converted to amino beads, and then to glutathione beads. We use standard practices with high concentrations of reagents to obtain a high glutathione site density on the beads. A high surface coverage of glutathione (GSH) enables robust capture of soluble GST fusion proteins [28]. The 12 Cyto-Plex™ carboxyl bead sets are coded with 12 graded intensities of far-red fluorescence when excited at a fixed wavelength, which does not interfere with fluorescein or phycoerythrin fluorescence detection. We suggest the use of a centrifuge with a swinging-bucket rotor and slow deceleration for ease of removing 90% of the supernatant, without disturbing the beads, during the many centrifugations in this synthesis. All reactions are at room temperature. While the protocol below is written for Cyto-Plex™ beads , we used an inexpensive (nonfluorescent) set of polystyrene carboxyl beads for pilot-testing our protocol for optimizing the synthetic conversion of carboxyl- to amino-functional groups on the beads, and subsequent coupling to glutathione (see Note 1).
?. [3.1 Synthesis of Glutathione Beads]
A bead set in its bottle is rocked gently on its side for , rotated ¼ turn and rocked again for , and continued until the beads are in a milky suspension. 15 s of immersion in a low-power ultrasonic bath can help the resuspension.
?. [3.1 Synthesis of Glutathione Beads]
Place  in a 0.65 mL centrifuge tube, add , mix gently with a pipette, and then allow the suspension to settle  to coat the beads and the tube with Tween-20, decreasing bead aggregation and adhesion to the tube (see Note 2).
[1% Tween-20]
[bead suspension]
?. [3.1 Synthesis of Glutathione Beads]
Remove all but ~10 μL of the supernatant, resuspend the beads, and give two standard washes:
?. [3.1 Synthesis of Glutathione Beads]
For a regular wash, add  to 10 μL of suspension, mix with a vortex mixer, centrifuge at , remove 100 μL of supernatant, and resuspend the remaining 10 μL of beads with a vortex mixer.
[pH 6 buffer]
Centrifuge: 5000 34, 2 min
This standard wash assures that a nominal factor of 10× dilution of the undesired solute is achieved. Resuspension in minimal buffer ensures equal exposure of all beads to the next reagent.
?. [3.1 Synthesis of Glutathione Beads]
Repeat the wash: For a regular wash, add  to 10 μL of suspension, mix with a vortex mixer, centrifuge at , remove 100 μL of supernatant, and resuspend the remaining 10 μL of beads with a vortex mixer.
[pH 6 buffer]
Centrifuge: 5000 34, 2 min
This standard wash assures that a nominal factor of 10× dilution of the undesired solute is achieved. Resuspension in minimal buffer ensures equal exposure of all beads to the next reagent.
?. [3.1 Synthesis of Glutathione Beads]
Weigh  and  into a microfuge tube, add , immediately dissolve by vortexing, add this to a bead set, and mix. Place the microfuge tube in a rotator with a horizontal axis of rotation for  to keep the beads in suspension, away from the tube lid and sides, while the site density of sNHS ester intermediate builds on the beads.
[EDAC]
[sNHS]
[pH 6 buffer]
?. [3.1 Synthesis of Glutathione Beads]
Centrifuge at , remove all but 10 μL of the supernatant, resuspend the beads, and then wash two times with , which will dilute the EDAC and sNHS while keeping the pH low and the sNHS ester intact.
Centrifuge: 5000 34, 2 min
[rinse solution]
?. [3.1 Synthesis of Glutathione Beads]
Resuspend the beads in , immediately add , mix, and rotate as in step 5 (place the microfuge tube in a rotator with a horizontal axis of rotation) for . Centrifuge at , remove all but 10 μL of supernatant, and resuspend the beads.
[pH 8.4 buffer]
[2 M 1,6 diaminohexane]
Centrifuge: 3000 34, 2 min
?. [3.1 Synthesis of Glutathione Beads]
Wash four times with pH 8.4 buffer and resuspend the amino beads into a total of 90 μL of pH 8.4 buffer:
?. [3.1 Synthesis of Glutathione Beads]
(Wash 1/4): Wash with pH 8.4 buffer.
?. [3.1 Synthesis of Glutathione Beads]
(Wash 2/4): Wash with pH 8.4 buffer.
?. [3.1 Synthesis of Glutathione Beads]
(Wash 3/4): Wash with pH 8.4 buffer.
?. [3.1 Synthesis of Glutathione Beads]
(Wash 4/4): Wash with pH 8.4 buffer.
?. [3.1 Synthesis of Glutathione Beads]
Resuspend the amino beads into a total of .
[ pH 8.4 buffer]
?. [3.1 Synthesis of Glutathione Beads]
We derivatize six sets of beads at a time and leave the six sets  at this stage. The amino site density can be measured in a pilot assay to ensure optimal conversion of carboxyl- to amino-terminal groups (see Note 3).
?. [3.1 Synthesis of Glutathione Beads]
Add , mix, rotate as in step 5 (place the microfuge tube in a rotator with a horizontal axis of rotation) for  while the site density of the crosslinker’s maleimide builds on the beads, centrifuge, and resuspend the beads in .
[200 mM sSMCC in DMSO]
10 µl
?. [3.1 Synthesis of Glutathione Beads]
Wash with  and resuspend to .
[pH 7 buffer]
[pH 7 buffer]
?. [3.1 Synthesis of Glutathione Beads]
Prepare and test the nitrogen-bubbling apparatus to give a slow series of bubbles (see Note 4).
?. [3.1 Synthesis of Glutathione Beads]
Add  and  and bubble nitrogen slowly through the suspension for  to remove most of the oxygen. Cap the tube and rotate it slowly for .
[100 mM EDTA]
[200 mM glutathione, pH 7]
?. [3.1 Synthesis of Glutathione Beads]
Centrifuge at , remove all but 10 μL of supernatant, and resuspend the glutathione beads. Wash beads four times in the storage buffer of your choice, reducing the concentration of glutathione from 20 mM to below 2 μM:
Centrifuge: 3000 34, 2 min
?. [3.1 Synthesis of Glutathione Beads]
Add  and  in the storage buffer to inhibit bacterial growth. Store at  at a concentration of 108 beads/mL. The beads have been stable for over 2 years. A portion of each bead set is diluted 10× in a storage buffer for ease of assay. Each assay uses 104 beads per target GTPase or 1 μL of diluted beads.
[EDTA]
[sodium azide]
4 °C
It is useful to quantify the number of GST-binding sites on the newly functionalized beads using commercial Quantum™ FITC MESF (see Fig. 2) or other methods [29, 30]. Assay the beads by incubating them in 25 nM GST-GFP for 30 min. Use 100 μM soluble glutathione to determine nonspecific binding of GST-GFP to the glutathione beads. Using Quantum™ FITC MESF beads, the glutathione beads synthesized as described support >1.0 million GST-binding sites. At high surface density (>1300 fluorophores/μm2), fluorophores on a bead surface undergo self-quenching with increasing site occupancy [31]. It is therefore likely that the 1.0 million sites determined by the calibration beads are a lower limit. However, this measure is useful for tracking the useful shelf life of the beads.
?. [3.1 Synthesis of Glutathione Beads]
(Wash 1/4): Wash beads in the storage buffer of your choice (reducing the concentration of glutathione from 20 mM to below 2 μM).
?. [3.1 Synthesis of Glutathione Beads]
(Wash 2/4): Wash beads in the storage buffer of your choice (reducing the concentration of glutathione from 20 mM to below 2 μM).
?. [3.1 Synthesis of Glutathione Beads]
(Wash 3/4): Wash beads in the storage buffer of your choice (reducing the concentration of glutathione from 20 mM to below 2 μM).
?. [3.1 Synthesis of Glutathione Beads]
(Wash 4/4): Wash beads in the storage buffer of your choice (reducing the concentration of glutathione from 20 mM to below 2 μM).