Cfi plan apo lambda 100x oil

Binding of HMM-GFP to actin filaments was observed as follows. G-actin was polymerized in NF-buffer (50 mM KCl, 2 mM MgCl 2 , 0.5 mM EGTA, 1 mM DTT, 20 mM PIPES, pH 6.5) containing 0.2 mM ATP for 2 h at 22°C. Actin filaments and Alexa 647 phalloidin (Invitrogen)
were mixed at a molar ratio of 20:1 and incubated overnight on ice. The surface of coverslips was covered with a positively charged lipid bilayer and was used to construct flow chambers as described previously (18) , except that the weight ratio of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC;
Avanti Polar Lipids, Alabaster, AL) and 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP;
Avanti Polar Lipids) was 17:3 (14) . Alexa 647 phalloidin-stabilized actin filaments diluted in NF-ATP buffer (NF buffer containing 0.5 µM ATP) were introduced into the flow chamber to loosely bind on the positively charged lipid monolayer. HMM-GFP and Rng2CHD diluted in NF-ATP buffer were then introduced to the flow chamber. Alternatively, NF-ATP buffer in the above procedures was replaced with NF buffer for the assays in the nucleotide-free state. Fluorescence of Alexa 647 and GFP was imaged with a fluorescence microscope (ECLIPSE E600, Nikon, Tokyo, Japan) equipped with an ARUGUS-HiSCA system (Hamamatsu Photonics, Hamamatsu, Japan). Images were captured using a 100x objective lens (CFI Plan Apo Lambda 100x Oil, NA 1.45; Nikon).