Figure 3

Analysis of hTERT TF purification by gel electrophoresis and Southwestern blotting. (A) Silver stained 1D Gel Electrophoresis using hTERT promoter trapping. Proteins from the eluate were separated on a 7.5% SDS polyacrylamide gel. Lanes consist of NE, flow through (FT), and elute (E). (B) Two-Dimensional PAGE. Eluate was subjected to 2DGE gel electrophoresis. The first dimension, isoelectric focusing, was achieved with a 7 cm, pH 3 10 IPG strip. The second dimension, separation by molecular weight, was done with 12% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Visualization was accomplished by silver staining. (C) DNA-Binding by Two-Dimensional Southwestern Blot. The 2-DE gel shown in Figure 1 was electroblotted onto a PVDF membrane and probed with the 2.0 nM radiolabeled hTERT promoter. Spots indicate the number of high affinity DNA-binding components of the hTERT complex that were purified by promoter trapping. In panels B and C, boxes with numbers show regions of the blot excised for further analysis.