Figure 1

Validation of monoclonal antibodies against RNA polymerase II. (A) Schematic of the work flow. P. falciparum samples were harvested every 8 h across the 48 h life-cycle at time points (TP) 1 through TP6. The same culture was used for triplicate ChIP-on-chip and transcriptome analyses. (B–D) Peptide competition assays. Monoclonal antibodies were raised against peptides corresponding to the unmodified heptad repeat (anti-CTD), the heptad repeat monophosphorylated at position 5 (αSer5-P) and diphosphorylated at positions 2 and 5 (αSer2/5-P). Each monoclonal antibody was pre-incubated with each of the three immunogenic peptides derived from the RNAPII CTD. Reactivity against the immunizing peptide was then tested by ELISA. The concentration of competing peptide in μg/ml is given to the right of each graph. (E) Parasite nuclear protein lysate from an asynchronous culture was immunoprecipitated (IP) with each of the three monoclonal antibodies raised in this study or a non-specific IgG control. The precipitates were separated by 6% SDS-PAGE and Western blot (W) performed with the indicated antibodies. The appropriately sized band for P. falciparum RPB1 (279 KDa) was observed with all three antibodies (arrowhead). Two panels are shown for antibody α-Ser2/5-P and represent the same Western blot following a moderate and more extensive (W*) series of washes. Note that the only significant specific band is of the correct molecular mass for RPB1. No specific bands are detected by the α-CTD and α-Ser2/5 antibodies following IP with control IgG. The commercial anti-CTD antibody 8WG16 detects a band of the correct molecular mass for P. falciparum RPB1 along with several faster running species. The positions of the marker bands in KDa are given to the right of the panels they describe.