Hume et al. (2014). Methods in enzymology on CD-ROM/Methods in enzymology. doi: 10.1016/b978-0-12-397924-7.00020-0
…e from freshly slaughtered animals, are chilled and kept on ice immediately after collection, and then processed within 2–3 h. Frozen brains are available from commer- cial sources (e.g., Pel-Freez Biologicals), but we find that results are far more reproducible using brains obtained from a local abattoir. Buffers Extraction buffer 20 mM HEPES, pH 7.4, 0.5 mM ATP, 0.5 mM DTT, 100 mM KCl, 2 mM M
Abstract
The WAVE regulatory complex (WRC) is critical to control of actin polymerization at the eukaryotic cell membrane. By reconstituting WAVE-dependent actin assembly on silica microspheres coated with phospholipid bilayers in mammalian brain extracts, we discovered that membrane recruitment and activation of WRC require the cooperative action of two mammalian GTPases, Arf and Rac. Here, we describe detailed methods to generate phospholipid-coated microspheres and porcine brain extract and outline conditions necessary to reconstitute WRC-dependent motility. In addition, we describe how to generate acylated recombinant GTPases, anchor them to lipid-coated microspheres, and reconstitute GTPase activation of WRC.