TITLE: 3.2 Production of a Cleared Cell Lysate
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 3.2 'Production of a Cleared Cell Lysate' 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]
?. [3.2 Production of a Cleared Cell Lysate]
Two days before an assay seed a 48-well plate with 20,000 target cells in 100 μL of culture medium per well, resulting in about 50,000 cells the next day. The rate of cell proliferation might vary based on cell type and conditions. The critical target is 50,000 cells at the start of an experiment.
?. [3.2 Production of a Cleared Cell Lysate]
Remove the culture medium and replace with . Specific inhibitors of signaling can be added to the cells for the desired amount of time depending on the requirements of the reader’s assay to establish proper inhibition before stimulation.
[serum-free medium]
?. [3.2 Production of a Cleared Cell Lysate]
After stimulating the cells (see Section 3.5 in Protocol "Applications of G-Trap Assay" for examples), chill the plate in an ice/water bath. Add  to each well with a 1 mL pipette and triturate the mixture gently to achieve homogenous lysis of the cells.
[cold 2× RIPA buffer]
?. [3.2 Production of a Cleared Cell Lysate]
Transfer the lysate into a 0.65 mL microfuge tube and centrifuge at . The 200 μL of cleared lysate is enough for triplicate assays using 50 μL for each test.
Centrifuge: 5000 34, 2 min