3.1 Synthesis of Glutathione Beads

Peter Simons, Virginie Bondu, Angela Wandinger-Ness, Tione Buranda

Published: 2021-09-03 DOI: 10.17504/protocols.io.bptqmnmw

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 3.1 'Synthesis of 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

3.1 Synthesis of Glutathione Beads

Note

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 ).

A bead set in its bottle is rocked gently on its side for 0h 2m 0s, rotated ¼ turn and rocked again for 0h 2m 0s, and continued until the beads are in a milky suspension. 15 s of immersion in a low-power ultrasonic bath can help the resuspension.

Place 4µL in a 0.65 mL centrifuge tube, add 400µL, 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 ).

Remove all but ~10 μL of the supernatant, resuspend the beads, and give two standard washes:

4.1.

For a regular wash, add 100µL to 10 μL of suspension, mix with a vortex mixer, centrifuge at 5000x g, remove 100 μL of supernatant, and resuspend the remaining 10 μL of beads with a vortex mixer.

Note

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.

4.2.

Repeat the wash: For a regular wash, add 100µL to 10 μL of suspension, mix with a vortex mixer, centrifuge at 5000x g, remove 100 μL of supernatant, and resuspend the remaining 10 μL of beads with a vortex mixer.

Note

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.

Weigh 4mg and 8mg into a microfuge tube, add 100µL, 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 0h 30m 0s 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.

Centrifuge at 5000x g, remove all but 10 μL of the supernatant, resuspend the beads, and then wash two times with 100µL, which will dilute the EDAC and sNHS while keeping the pH low and the sNHS ester intact.

Resuspend the beads in 180µL, immediately add 20µL, mix, and rotate as in step 5 (place the microfuge tube in a rotator with a horizontal axis of rotation) for 0h 30m 0s. Centrifuge at 3000x g, remove all but 10 μL of supernatant, and resuspend the 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:

8.1.

(Wash 1/4): Wash with pH 8.4 buffer.

8.2.

(Wash 2/4): Wash with pH 8.4 buffer.

8.3.

(Wash 3/4): Wash with pH 8.4 buffer.

8.4.

(Wash 4/4): Wash with pH 8.4 buffer.

8.5.

Resuspend the amino beads into a total of 90µL.

8.6.

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 ).

Add 10µL, mix, rotate as in step 5 (place the microfuge tube in a rotator with a horizontal axis of rotation) for 0h 30m 0s while the site density of the crosslinker’s maleimide builds on the beads, centrifuge, and resuspend the beads in 10µL.

10.

Wash with 100µL and resuspend to 360µL.

11.

Prepare and test the nitrogen-bubbling apparatus to give a slow series of bubbles ( see Note 4 ).

12.

Add 2µL and 20µL and bubble nitrogen slowly through the suspension for 0h 2m 0s to remove most of the oxygen. Cap the tube and rotate it slowly for 0h 30m 0s.

13.

Centrifuge at 3000x g, 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:

13.1.

(Wash 1/4): Wash beads in the storage buffer of your choice (reducing the concentration of glutathione from 20 mM to below 2 μM).

13.2.

(Wash 2/4): Wash beads in the storage buffer of your choice (reducing the concentration of glutathione from 20 mM to below 2 μM).

13.3.

(Wash 3/4): Wash beads in the storage buffer of your choice (reducing the concentration of glutathione from 20 mM to below 2 μM).

13.4.

(Wash 4/4): Wash beads in the storage buffer of your choice (reducing the concentration of glutathione from 20 mM to below 2 μM).

14.

Add 1millimolar (mM) and 0.02% (w/v) in the storage buffer to inhibit bacterial growth. Store at 4°C at a concentration of 10⁸beads/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 10⁴beads per target GTPase or 1 μL of diluted beads.

Note

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.

Fig. 2Single-analyte assay for RILP: GTP·Rab7 captured on beads. Fluorescently labeled detection antibody added to lysis buffer is used to assess nonspecific binding of the antibody to beads. Flow cytometry histograms of RILP-RBD effector beads incubated at 4 °C with resting HeLa cell lysates or with EGF -stimulated HeLa cell lysates show increased Rab7-GTP bound, the levels of which can be quantified using commercial standard calibration beads (Quantum™ FITC MESF). Quantum™ FITC MESF beads comprise five sets of distinct bead populations. Each bead population is distinguished by a discrete number of doped fluorophores of known calibration. The average fluorophores/bead on each bead population is shown on the x-axis. The calibration beads are used to quantify the occupancy of Rab7-specific antibodies on RILP-effector beads. After correcting for nonspecific binding, 7.1 ± 1.2 × 103 Rab7-GTP molecules/bead were recovered in resting cell lysates, and 6.7 ± 0.3 × 104 Rab7-GTP molecules/bead were retrieved in EGF-stimulated cell lysates [16]

3.1 Synthesis of Glutathione Beads

Abstract

Steps

3.1 Synthesis of Glutathione Beads

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