Rabbit igg antibodies

An equivalent of 1 × 10⁹ 3D7+empty vector or 3D7+PfAlba1-Ty1 parasites were grown to 28–30 h trophozoite stages in RPMI complete medium supplemented with 5 μg/ml BS, infected red blood cells harvested, and free parasites collected by saponin lysis. The resulting parasite pellet was lysed under non-denaturing conditions in the presence of RNAsin (Promega) and subjected to immunoprecipitation analysis with rabbit anti-Ty1 antibodies or rabbit IgG antibodies (Sigma). IPed RNA was directly used to prepare strand-specific RNA-seq libraries (see below), without poly(A) enrichment. A minimum of two biological replicates was sequenced for each experimental and control immunoprecipitation. See Supplementary Materials and Methods in Additional file 1 for more details.

An affinity purification protocol optimized to detect transient interactions was used (43–45 (link)). Briefly, yeast cells were collected, suspended in resuspension buffer, frozen in liquid nitrogen and then lysed in solid phase by cryo-milling on a MM 400 mixer (Retsch's) and stored at −80°C until needed. The resuspension buffer consisted of: 1.2% PVP-40, 20 mM HEPES pH 7.4, 1:100 Sigma protease inhibitor cocktail, and 1:100 solution P (2 mg pepstatin A and 90 mg PMSF in 5 ml ethanol) and 1 mM DTT. The extract was resuspended in RNP buffer (20 mM HEPES pH 7.4, 110 mM KOAc, 0.5% Triton X-100, 0.1% Tween-20, 1:5000 Ambion SuperRNAsin, 1:5000 Sigma antifoam, 1:100 solution P, 150 mM NaCl) and incubated with prepared magnetic beads with rabbit IgG (Dynal) for 1 h at 4°C. The beads were collected using a magnet, washed five times, and eluted twice for 20 minutes at RT under denaturing conditions (500 mM NH₄OH/0.5 mM EDTA). Eluates were then lyophilized for LC–MS/MS analysis. For coated magnetic beads preparation, M280 Dynabeads Sheep anti-rabbit IgG (Dynal, Invitrogen) were saturated with rabbit IgG antibodies (Sigma) for 1 h at +4°C as recommended by the manufacturer. Magnetic beads were then washed extensively and rabbit IgG cross-linked using glutaraldehyde as described previously (45 (link)).