Figure 5. Direct DNA-binding affinity comparison for TERT complexes in bulk purifications.
(A) Schematic for detection of Cy5-labeled DNA binding to biotinylated TERT complexes. (B) SDS-PAGE analysis of MCP-TERT complexes labeled using CoA-Cy5. Cy5-labeled MCP-TERT proteolysis products that retain the N-terminal F-MCP tag and are enriched in the 293T-F purification are schematized in comparison to full-length TERT. (C) Concentration dependence of Cy5-labeled DNA retention by slide-anchored TERT complexes across a titration of 0.3, 1, 3, 10, and 30 nM DNA. Spot count per field was normalized to the 30 nM DNA quantification for each sample. Error bars represent standard error of the mean of spot counts of five fields per sample per DNA concentration. (D) Graph of the change in Cy5-labeled DNA spot count comparing assays of 30 vs 100 nM DNA, normalized to the 30 nM DNA quantifications for each sample. Error bars represent standard error of the mean of spot counts of five fields per sample per DNA concentration.