cells were resuspended at 1 2 u00d7 10 7 cells ml in 100 mm nacl 20 mm hepes ph 7 5 and lyzed by adding 2 w v sodium dodecylsulfate followed by incubation at 95 u00b0 c for 5 min threads were sedimented at 5 000 u00d7 g followed by washing with 8 m urea and water bidest purified filaments were resuspended in water bidest and incubated for 3 min on freshly cleaved mica afm or loaded on thermanox u00ae plastic cover slips nalgene nunc sem for afm samples were rinsed with water bidest four times and air dried prior to contact mode imaging using a multimode spm veeco for sem samples were air dried after removal of the solution vacuum coated with a gold layer and analyzed with a jsm 5900lv jeol ltd at 20 kv for tem jeol ltd analysis filaments were adsorbed onto formvar coated grids and negatively stained with uranyl acetate for immunostaining fibers were incubated with mouse anti his 6 antibodies followed by labeling with 18 nm gold conjugated goat anti mouse igg to rule out possible influences of the his 6 tag on filament formation t7 tagged adf 4 was synthesized in sf9 cells the filament formation of t7 tagged adf 4 was apparently indistinguishable to that of his 6 tagged.