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2, 1 mM EDTA, glycerol (20 μl/ml), 300 mM potassium acetate, 1 mM dithiothreitol (DTT)] containing 10% lipid emulsion or 0.2% octyl glucoside. Results obtained with octyl glucoside or lipids were identical; the experiments shown use lipid emulsions. [Lipid emulsions were prepared by hydrating a dried mixture of 2 mg of dipalmitoleoyl-phosphatidylethanolamine and 3 mg of dipalmitoleoyl-phosphatidylcholine (both from Avanti Polar Lipids) in 1 ml of BB and sonicating on ice with a probe sonicator for 4 × 30 s, followed by centrifugation for 5 min at 15,000g; the supernatant was used.] Where indicated, GMP-PNP or guanosine 5′-O-(2′-thiotriphosphate) (GDP-β-S) was present at 170 μM. The total reaction volume was 100 μl. After 90 min of rotation at 4°C, the reaction mixture was added to 15 μl of prewashed GSHA beads, and the incubation was continued for 30 min more. Beads were washed 3x with BB and 1x with 20 mM Na Hepes, pH 6.8, and then heated to 65°C for 10 min in SDS sample buffer. Eluted proteins were analyzed by SDS gel electrophoresis and Coomassie Blue R-250 staining.
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