Fig. 2. In vitro filament assembly properties of variant keratins. (A) SDS polyacrylamide gel electrophoresis plus Coomassie Blue staining to show (left to right) molecular weight markers (Mr in kDa as indicated), recombinant K8(wt) and K18(wt), respectively. (B) Coomassie Blue-stained SDS-PAGE of samples from a sedimentation assay of K8(wt), K8(G62C), K8(I63V) or K8(K464N) each with K18(wt), polymerized in vitro under reducing conditions. `P' denotes pelleted (i.e. filamentous) material; `S' denotes material recovered from the supernatant. K8(wt) and K18(wt) readily assemble into pelletable polymers, whereas the mutant K8 forms smaller amounts of pelleted material in the sedimentation assay. (C) Coomassie Blue-stained SDS-PAGE of a sedimentation assay of K8(wt) with K18(wt) or K18(S230T), polymerized in vitro under reducing conditions: K18(S230T) mutant behaves as wild type. (D) Electron micrograph showing long, uniform filaments formed by in vitro polymerization of K8(wt) with K18(wt). Polymerization of wild-type K18 with (E): K8(G62C), (F): K8(I63V) or (G): K8(K464N) produces shorter, less uniform filaments and more small particulate aggregates. (H) K18(S230T) forms filaments with K8 (wild type) that are similar to those formed by wild-type K18. Bars, 200 nm.