Tandem Affinity Purification of Trypanosome Proteins

For expression of N-terminal tandem affinity purification (TAP) tagged proteins, fragments of ZC3H41 (Tb927.11.1980) or Z41AP (Tb927.7.7460) open reading frames (ORFs) were cloned into p2676 [36 (link)] to yield pGR307 and pGR315, respectively. These plasmids were transfected into procyclic 449 cells and selected in the presence of 1 µg/ml puromycin. Oligonucleotides used for cloning are described in Additional file 1. Detection of TAP-tagged proteins was carried out by immunoblot analysis using Papanicolaou reagent (Sigma) or an anti-protein A antibody (Sigma), whereas 4xTy-tagged protein levels were monitored using a BB2 monoclonal antiserum [37 (link)]. Other antisera used were anti-α-tubulin (clone B-5-1-2; Sigma), anti-p34/p37 (NRBD1/2; [38 (link)]), anti-P0 [39 (link)], anti-S9 [unpublished; a kind gift from Christine Clayton (Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg)], anti-DRBD3 [40 (link)], anti-RRP4 [6 (link)] and anti-puromycin (clone 12D10; Sigma). To prepare total cell extracts for immunoblot analysis, cells were harvested from logarithmic cultures, lysed in Laemmli buffer, boiled, and loaded directly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (1–2 × 10⁶ cells/lane). Proteins were visualized using either chemiluminescence or an Odyssey CLx Near-Infrared Fluorescence Imaging System (LI-COR Biosciences).