Antibody Characterization and Validation Protocol

In this study, we used the following antibodies (also specified in relevant Methods sections above): Monoclonal antibodies: rat anti-HA (Roche 3F10, Cat.: 11867423001, Lot: 47877600), mouse anti-FLAG (Sigma M2, Cat.: F1804, Lot: SZCD3524), or mouse monoclonal antibodies produced in-house: anti-HA (12CA5), anti-FLAG 9H1. Polyclonal antibodies against RhopH2, RhopH3 and Clag3 were generated in rabbits by GenScript against the following antigens: RhopH3 T731-Y829, RhopH2 L20-S1378 and Clag3 K1277-H1417 and verified using indirect ELISA by the manufacturer. Rabbit anti-RON4 serum was used as published before^{34 (link)}. The following secondary Alexa 488/594 fluorophores from Life Technologies were used: chicken anti-mouse 594 (Cat.: A21201, Lot: 42099 A), donkey anti-rat 488 (Cat.: A21208, Lot: 2310102), chicken anti-rabbit 594 (Cat.: A21442, Lot: 2110863). The following secondary HRP-conjugated antibodies were used: goat a-rat (Southern Biotech, Cat.: 3030-05, Lot: G2512-M748B), goat α-mouse (Merck Millipore, Cat: AP124P), goat a-rabbit (Merck Millipore, Cat: AP187P).

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Pasternak M., Verhoef J.M., Wong W., Triglia T., Mlodzianoski M.J., Geoghegan N., Evelyn C., Wardak A.Z., Rogers K, & Cowman A.F. (2022). RhopH2 and RhopH3 export enables assembly of the RhopH complex on P. falciparum-infected erythrocyte membranes. Communications Biology, 5, 333.

Publication 2022

mouse anti-FLAG donkey anti-rat 488 goat a-rat goat α-mouse goat a-rabbit

Alexa 594 Antibodies Antigens Chicken Donkey Elisa Goat Monoclonal antibodies Mouse Rabbit Rabbits Serum

Corresponding Organization :

Other organizations : Walter and Eliza Hall Institute of Medical Research, University of Melbourne

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Variable analysis

independent variables

Monoclonal antibodies: rat anti-HA, mouse anti-FLAG, anti-HA (12CA5), anti-FLAG 9H1
Polyclonal antibodies against RhopH2, RhopH3 and Clag3

dependent variables

Not explicitly mentioned

control variables

Rabbit anti-RON4 serum

positive controls

Rabbit anti-RON4 serum

negative controls

Not explicitly mentioned

Annotations

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