Pyrene-actin polymerization assays
This protocol is extracted from research article:
The structure of the actin filament uncapping complex mediated by twinfilin
Sci Adv, Jan 27, 2021; DOI: 10.1126/sciadv.abd5271

Pyrene-actin polymerization assays to monitor filament uncapping were performed in 100-μl reactions containing 2 μM rabbit skeletal muscle G-actin (10% pyrene–labeled), 0.5 μM F-actin seeds, 2.8 μM profilin (to prevent pointed-end polymerization), 100 nM CP, and variable concentrations of the test proteins. In all pyrene-actin polymerization assays, the final concentrations of twinfilin, CP, CARMIL, V-1, MBP-tagged tail, and MBP were set at 1 μM, 100 nM, 250 nM, 5 μM, 10 μM, and 10 μM, respectively. The components were mixed in buffer A [2 mM tris-HCl (pH 7.5), 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN3, and 0.1 mM CaCl2] to a final volume of 70 μl in a black flat-bottom 96-well plate (Corning).

To prepare actin filament seed stocks, 5 μM G-actin was polymerized for 1 hour at room temperature, after which the filaments were mechanically sheared by repeatedly passing the F-actin solution through a 0.7-mm-diameter needle for 1 min. Thereafter, 10 μl of the F-actin seed stock was mixed with 10 μl of 1 μM CP and left for 5 min to allow barbed-end capping of the F-actin seeds. As a control, the F-actin seed stock was mixed with buffer A, without CP. Then, 20 μl of this precapped F-seed stock, or control, was added to pyrene-actin polymerization mixture and actin polymerization initiated by addition of 10 μl of 10× KMEI buffer (500 mM KCl, 10 mM MgCl2, 10 mM EGTA, and 100 mM imidazole, pH 7.4) in a total reaction volume of 100 μl. The pyrene fluorescence intensities were monitored immediately on a Safire2 fluorimeter plate reader (Tecan) with excitation and emission wavelengths set at 365 and 407 nm, respectively.

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