Abstract
This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could also be used to detect antigens within the parasite if the sample is appropriately fixed and permeabilised before addition of the relevant antibody. While access of the antibody will avoid the detection problems often seen with post-embedding methods, this procedure will produce comparably poorer morphology.
Keywords: Parasitology, Malaria, Plasmodium falciparum, Electron Microscopy
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
We gratefully acknowledge the work from the Tilley lab that developed the method for pre-embedding EM in selectively permeabilsed P. falciparum-infected cells that forms the basis for the procedures described here: Jackson et al. (2007). The Percoll gradient is a modified version of the protocol from Aley et al. (1986). The Percoll purification is described in more detail in the Bio-protocol 'Preparation of parasite protein extracts and Western blot analysis' (Procedure I.2.a-e).
References
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