196 G.W. Gitau et al. Fig. 1 (continued) Size exclusion chromatography and co-immunoprecipitation analysis Size exclusion chromatography was performed to investigate whether PfHop, PfHsp70, PfHsp90 and PfJ4, a potential Hsp40 co-chaperone of PfHsp70 (Pesce et al. 2008) existed as a complex in parasite cells. The eluted fractions obtained were subjected to Western analysis using anti-PfHop, anti- PfHsp70, anti-PfHsp90 and anti-PfJ4 antibodies (Pesce et al. 2008; Fig. 5a). PfHsp90, PfHsp70 and PfHop all appeared in an apparent fraction of approximately 400 kDa in size. Several possibilities may underpin the observed findings. One interpretation is that all the three proteins are in a common complex. It is also possible that PfHsp90, PfHsp70 and PfHop may have been eluted in independent complexes that were apparently of similar sizes, thus eluting in the same fraction. Alternatively, this could represent independent homo-oligomers that approximated each other in size, and thus were eluted in the same fractions, but were not necessarily associated with one another. A previous study suggested that a type II (Cheetham and Caplan 1998) Hsp40 protein, PfJ4 associated with PfHsp70 (Pesce et al. 2008). However, it seemed that PfJ4 was not part of the putative complex made up of PfHop, PfHsp70 and PfHsp90 (Fig. 5a). Formation of the Hsp70–Hop–Hsp90 complex is known to be nucleotide sensitive. As a result, we sought to establish whether PfHop, PfHsp70 and PfHsp90 association was nucleotide-sensitive. A co-immunoprecipitation study was conducted using anti-PfHsp70 antibodies and passing parasite lysate through the column. In one instance, 5 mM ATP was added to the parasite lysate before conducting the immunoprecipitation (lane ‘+ATP’, Fig. 5b) and the