Figure 9. Separation of intermediate cyclin B and NH2-terminal fragment by gel chromatography. (A) Sephadex G-50 column chromatography. The 35S-labeled cyclin Δ0 was produced in vitro in rabbit reticulocyte lysate. Digestion of 35S-labeled cyclin Δ0 was performed for 60 min at room temperature with (•) or without (○) 26S proteasome. Samples were then separated on Sephadex G-50 column (1.0 × 19.0 cm) in 100 mM Tris-HCl, 5 mM MgCl2, pH 7.6. Fractions of 0.5 ml were collected. Arrows indicate the eluted positions of molecular weight standards as follows: 1, bovine serum albumin; 2, myoglobin; 3, ubiquitin; 4, total column volume (Vt). (B) SDS-PAGE analysis of gel chromatography fractions. Sephadex G-50 column chromatography fractions from 26S proteasome-treated cyclin Δ0 and untreated (Control) were separated by SDS-PAGE (15% gel) followed by autoradiography on Imaging plates (Fuji Film). The positions of the digested cyclin B is indicated by an asterisk, and the positions of NH2-terminal portion of cyclin B is indicated by an arrowhead.
Image published in: Tokumoto T et al. (1997)
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