FIG. 2.

Direct affinity purification of VP22-associated proteins from uninfected HeLa cell extracts. (A) Cell extracts (2 × 10¹⁰ cells) were passed over a VP22 affinity column, constructed as described in Materials and Methods. Lanes: M, molecular size standard; In, input (2 × 10⁴ cell equivalents); Un, unbound (2 × 10⁴ cell equivalents); El, 600 mM NaCl elution fraction (10⁸ cell equivalents). Proteins were separated by SDS-PAGE and detected by silver staining. While the vast majority of proteins were not retained on the column, a high degree of enrichment of four species was observed (numbers indicate molecular mass in kilodaltons). (B) The elution fraction was transferred to nitrocellulose and reacted with anti-nonmuscle myosin II antibody. Elution fraction arrows indicate a large species of approximately 225 kDa in the bound fraction eluting at 600 mM NaCl.