VAR2CSA Ectodomain Labeling in Plasmodium falciparum Infected Red Blood Cells and Analysis via Flow Cytometry
- Author(s)
- Carmo, OMS; Dixon, MWA;
- Details
- Publication Year 2023-08-05,Volume 13,Issue #15,Page e4725
- Journal Title
- Bio Protocol
- Abstract
- Presentation of the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected red blood cells (RBCs) underpins the malaria parasite's pathogenicity. The transport of EMP1 to the RBC surface is facilitated by a parasite-derived trafficking system, in which over 500 parasite proteins are exported into the host cell cytoplasm. To understand how genetic ablation of selected exported proteins affects EMP1 transport, several EMP1 surface presentation assays have been developed, including: 1) trypsinization of surface-exposed EMP1 and analysis by SDS-PAGE and immunoblotting; and 2) infected RBC binding assays, to determine binding efficiency to immobilized ligand under physiological flow conditions. Here, we describe a third EMP1 surface presentation assay, where antibodies to the ectodomain of EMP1 and flow cytometry are used to quantify surface-exposed EMP1 in live cells. The advantages of this assay include higher throughput capacity and data better suited for robust quantitative analysis. This protocol can also be applied to other cellular contexts where an antibody can be developed for the ectodomain of the protein of interest.
- Keywords
- Flow cytometry; Live cell; Malaria; Protein transport; Surface proteins
- Research Division(s)
- Infectious Diseases And Immune Defence
- PubMed ID
- 37575395
- Publisher's Version
- https://doi.org/10.21769/BioProtoc.4725
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2023-08-16 02:59:26
Last Modified: 2023-12-11 03:08:34