FIG. 2.

PTP purification of TbXPD. (A) Schematic depiction of the two TbXPD alleles in the clonal cell line TbX1. One allele was replaced by the hygromycin phosphotransferase gene (HYG-R), while the vector pXPD-PTP-NEO was inserted into the second allele. The TbXPD coding region is represented by open boxes, the PTP tag by a black box, resistance marker coding regions by striped boxes, and introduced gene flanks for RNA processing signals by smaller gray boxes. (B) Immunoblot monitoring of the XPD-PTP purification. Aliquots of input material, the flowthrough of the IgG columns (FT-IgG), the TEV protease eluate (Elu TEV), the flowthrough of the anti-ProtC column (FT-ProtC), and final EGTA (Elu EGTA) and peptide (Elu PEP) elutions were separated on a 10% SDS-PAGE and detected with a monoclonal antibody directed against the ProtC epitope. Values followed by × indicate relative amounts of each fraction analyzed. Marker sizes are depicted on the left, and the size difference between the full-length XPD-PTP and the TEV protease-cleaved XPD-P is indicated on the right. (C) Total eluate of the TbXPD-PTP tandem affinity purification was separated on a 10 to 20% SDS-polyacrylamide gradient gel and stained with Coomassie blue. For comparison, 0.002% of the input material and 5% of the TEV protease eluate (Elu TEV) were loaded. The four unambiguously identified proteins are specified on the right, whereas protein marker sizes are listed on the left.