Merck

Differential Fractionation of Erythrocytes Infected by Plasmodium berghei.

Bio-protocol (2021-03-05)
Bénédicte Gnangnon, Véronique Peucelle, Christine Pierrot
RESUMO

The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected erythrocyte. The task remains delicate despite the use of enzymes or detergents theoretically capable of degrading specific membranes inside the infected cell. The remit of this protocol is to propose a method to isolate the erythrocyte cytosol and ghosts from the other compartments of the infected cell via a percoll gradient. Also, the lysis of the erythrocyte membrane is done using equinatoxin II, which has proven to be more effective at erythrocyte lysis regardless of the cell infection status, compared to the commonly used streptolysin. The parasitophorous vacuole (PV) content is collected after saponin lysis, before recovering membrane and parasite cytosol proteins by Triton X-100 lysis. The lysates thus obtained are analyzed by Western blot to assess the accuracy of the various extraction steps. This protocol allows the separation of the host compartment from the parasite compartments (PV and parasite), leading to potential studies of host proteins as well as parasite proteins exported to the host cell.

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Sigma-Aldrich
Ethylenediaminetetraacetic acid calcium disodium salt
Sigma-Aldrich
Saponin from quillaja bark, Sapogenin content ≥10 %
Sigma-Aldrich
D-Sorbitol, ≥98%
Sigma-Aldrich
Magnesium sulfate, anhydrous, ReagentPlus®, ≥99.5%