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Actin depolymerization assay

SS Souvarish Sarkar
FB Farah Bardai
AO Abby L. Olsen
KL Kelly M. Lohr
YZ Ying-Yi Zhang
MF Mel B. Feany
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Actin depolymerization assays were performed using the fluorescent form of the Actin Polymerization Biochem Kit from Cytoskeleton Inc. (Cat. #BK003), as described by the manufacturer. Pyrene-labeled actin was polymerized at room temperature for 1 h in actin polymerization buffer (containing 2 mM MgCl2 and 2 mM ATP) and then mixed either with buffer or with human recombinant LRRK2, LRRK2-G2019S, or a 1:1 mixture of the two. Actin was used at a final concentration of 4 µM and the LRRK2 proteins at 1 nM. The samples were read every 30 s in a plate reader at the excitation/emission wavelengths of 350/410 for 1 h. The experiment was repeated three times with two technical replicate each time per sample. The values were normalized to 0 at the starting time. Each data point represents the mean of three separate experiments.

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