The mAbs specific for TgAMA1 were CL22 [18 (link)] and B3.90 [19 (link)]. For immunoprecipitation of TgAMA1, these monoclonal antibodies were bound and coupled using dimethyl pimelimidate to fastflow Protein G sepharose (Amersham Biosciences, Little Chalfont, United Kingdom) [59 ].
Tachyzoite lysates were generated by extracting 5 × 109 extracellular parasites in 10 ml of TEN buffer (50 mM Tris [pH 8.0], 5 mM EDTA, 150 mM NaCl) containing RIPA detergents [1% NP40, 0.5% Deoxycholate, 0.01% SDS, and Complete protease inhibitor, (Roche Diagnostics, Mannheim, Germany)] on ice for 30 min and removing insoluble material by centrifugation at 3,000 × g for 20 min.
These extracts were incubated with the indicated antibodies coupled to protein G sepharose for 4 hr at 4 °C, followed by three washes (15-min each) in RIPA buffer and three washes in Tris-buffered saline (TBS; 50 mM Tris [pH 8.0], 150 mM NaCl). Bound polypeptides were eluted with 0.1 M triethylamine [pH 11.5] (using five successive elutions), and lyophilized to concentrate the eluate and remove the triethylamine. The 106 parasite equivalents were fractionated by SDS-PAGE for immunoblot analysis, or, similarly, 108 parasites for Coomassie brilliant blue staining.
Tachyzoite lysates were generated by extracting 5 × 109 extracellular parasites in 10 ml of TEN buffer (50 mM Tris [pH 8.0], 5 mM EDTA, 150 mM NaCl) containing RIPA detergents [1% NP40, 0.5% Deoxycholate, 0.01% SDS, and Complete protease inhibitor, (Roche Diagnostics, Mannheim, Germany)] on ice for 30 min and removing insoluble material by centrifugation at 3,000 × g for 20 min.
These extracts were incubated with the indicated antibodies coupled to protein G sepharose for 4 hr at 4 °C, followed by three washes (15-min each) in RIPA buffer and three washes in Tris-buffered saline (TBS; 50 mM Tris [pH 8.0], 150 mM NaCl). Bound polypeptides were eluted with 0.1 M triethylamine [pH 11.5] (using five successive elutions), and lyophilized to concentrate the eluate and remove the triethylamine. The 106 parasite equivalents were fractionated by SDS-PAGE for immunoblot analysis, or, similarly, 108 parasites for Coomassie brilliant blue staining.
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