Fig. 2.

Identification of disassembly factor as the CCT/TRiC chaperonin. (A) Final purification of disassembly factor from HeLa cells by ATP affinity chromatography. Partially purified disassembly factor from pooled fractions 17–21 of the gel filtration column (Fig. S3) was applied to 0.5 mL of 6-AH-ATP affinity resin (Jena Biosciences). ATP affinity chromatography was performed according to instructions of the manufacturer, except that additional 10 mM MgCl₂ was added to the wash buffer, and 10 mM ATP with 10 mM MgCl₂ was added to the elution buffer. Eight consecutive elutions of 1 mL were collected and concentrated fourfold by ultrafiltration. Disassembly of MCC (Materials and Methods) was assayed in samples of 0.5-μL eluates and samples of 1 μL were blotted for TCP-1α and HSP70. (B) Silver staining of ATP affinity chromatography eluates of disassembly factor. Samples of 5 μL of the indicated ATP-affinity eluates (Fig. 2A) were electrophoresed on 4–12% Bis-Tris protein gels (Nupage, Invitrogen). The marked protein bands (encircled) were excised and subjected to tryptic digestion and mass spectrometric analysis. Identifications of proteins are shown on the Right, and electrophoretic migration positions of marker proteins (in kilodaltons) are shown on the Left.