Pyrene assay

SW Shuaijun Wang
AC Alvaro H. Crevenna
IU Ilke Ugur
AM Antoine Marion
IA Iris Antes
UK Uli Kazmaier
MH Maria Hoyer
DL Don C. Lamb
FG Florian Gegenfurtner
ZK Zane Kliesmete
CZ Christoph Ziegenhain
WE Wolfgang Enard
AV Angelika Vollmar
SZ Stefan Zahler
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Pyrene actin was purchased from Hypermol (Bielefeld, Germany) and diluted to a 1 mg/mL (24 μM) stock solution. Before use, spontaneously formed actin aggregates were removed by ultracentrifugation for 1 h at 40,000 rpm and 4 °C. 50 μL samples for the pyrene assay consisted of: 30 μL H2O, 10 μL 10 mM MgCl2 or 250 mM KCl, 5 μL F-actin Buffer (100 mM Imidazole-Cl pH 7.4, 10 mM ATP, Hypermol, Germany) as well as 5 μL DMSO (containing the indicated concentrations of MiuA). 10 μL pyrene actin (end concentration 1 µM) were rapidly added to start polymerization. Pyrene fluorescence was monitored at 400 nm every 20 s over 1 h in a 96-well fluorescence plate reader (Tecan) with 360 nm excitation.

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