Journal: Molecular and Cellular Biology
Article Title: Molecular Architecture of the Human Prp19/CDC5L Complex ▿Molecular Architecture of the Human Prp19/CDC5L Complex ▿ †
Figure Lengend Snippet: The native human Prp19/CDC5L complex contains four copies of hPrp19. (A) Determination of the protein stoichiometry by fluorescence staining. Four different amounts of hPrp19/CDC5L complex proteins (as indicated above each lane) were separated by SDS gel electrophoresis and stained with Sypro-Ruby. The fluorescence was detected by fluorescence imaging (FLA-7000; Fujifilm). (B) Determination of the protein stoichiometry by radioactive labeling. Denatured proteins (0.5 to 12.5 μg, as indicated) were labeled with [ 14 C]iodoacetamide, separated by SDS-PAGE and detected by autoradiography. (C) Band intensities obtained after Sypro-Ruby staining and [ 14 C]iodoacetamide labeling were quantified using Fuji Image Gauge v4.22 and normalized against the CDC5L signal. (D) Continuous size distribution [C (S) ] analysis of the hPrp19/CDC5L complex. Four different concentrations of the complex were analyzed (optical densities at 280 nm [OD 280 ] of 1.2, 0.6, 0.3, and 0.15) as indicated in the figure. The calculated C (S) is plotted against the sedimentation coefficient (S). The S values in the panel are corrected according to standard conditions [S (20,w) ]. (E) Multispeed sedimentation equilibrium analysis of the hPrp19/CDC5L complex at two different rotor speeds (gray, 5,500 rpm; black, 3,500 rpm). The OD 280 values along the cell were recorded and are shown in the upper panel. The lower panel shows the residuals between the fit (straight line in the upper panel) and the raw data (dots in the upper panel).
Article Snippet: Subsequently, the pH was increased to 9.0 with 100 mM Tris-HCl buffer, and [14 C]iodoacetamide (56 mCi/mmol; GE Healthcare) was added to a concentration of 50 mM.
Techniques: Fluorescence, Staining, SDS-Gel, Electrophoresis, Imaging, Labeling, SDS Page, Autoradiography, Sedimentation