Full-length Gag protein had been produced and purified as described previously (10). SLBs for Gag lipid clustering measurements were prepared from 30-nm small unilamellar vesicles (SUVs) with the following: DOPC, 68%; PS, 30%; PI(4,5)P2, 1.9%; and ATTO647N-PI(4,5)P2, 0.1% mol at 0.1 mg/ml in a citrate buffer [10 mM Na citrate, 100 mM NaCl, and 0.5 mM EGTA (pH 4.6)]. The SUVs were then spread for 45 min at 37°C on coverslips pretreated by sonication in 5% SDS solution and incubation for 30 min in freshly made piranha solution (H2SO4:H202 2:1 vol). After disposition of the SLBs, scanning FCS measurements were performed and again 15 min after Gag was added at 1 μM final concentration.

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