Generating Transgenic P. falciparum Lines
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Corresponding Organization :
Other organizations : Walter and Eliza Hall Institute of Medical Research, Deakin University, Burnet Institute
Protocol cited in 16 other protocols
Variable analysis
- Transfection of P. falciparum strain 3D7 with plasmids pPTEX150-HA/Str3' or pHSP101-HA/Str3'
- Immunoprecipitated proteins identified by LC-MS/MS or Western blot analysis
- Localization of proteins observed by immunofluorescence analysis
- Repeated drug cycling in the presence and absence of WR99210 to obtain parasites containing integrated forms of the plasmid
- Permeabilization of erythrocytes with tetanolysin or cross-linking with 2mM DSP followed by saponin lysis for immunoprecipitation experiments
- Parasite lysates generated by incubation on ice in 1% (v/v) Triton-X 100 or RIPA buffer
- Fixation of parasites with ice-cold 90% acetone/10 % methanol for immunofluorescence analysis
- Positive control: Recombinant hexa-his fusion proteins derived from either PTEX150 (amino acids 181-236) or HSP101 (amino acids 68-170) used to raise antibodies
- Negative control: Not explicitly mentioned
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