Parasite Protein Western Blot Assay

Full parasite protein western blot samples were collected by lysing RBCs with 0.1% saponin and boiling protein in Loading Buffer (50mM Tris-Cl pH 8.0, 20% SDS, 1% Bromophenol Blue). Fractionated parasite protein was prepared as described in [95 (link)]. Blots were performed as described [19 (link)]. Primary antibodies used were: 1/1000 rat ant-HA (Roche 3F10), 1/1000 mouse anti-GFP (Roche), 1/3000 rabbit anti-aldolase conjugated to HRP (Abcam ab38905), or 1/3000 mouse anti-H3 (Abcam ab10799). Secondary antibody concentrations used were 1/3000 goat anti-rat HRP conjugate (Millipore), 1/3000 goat anti-mouse HRP conjugate, or 1/10,000 (Pierce) goat anti-rabbit HRP conjugate (Millipore). ECL reagent (Pierce) was used to detect HRP signal. Blots were exposed to autoradiography film (VWR) and visualized using an autoradiography developer.

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Russell T.J., De Silva E.K., Crowley V.M., Shaw-Saliba K., Dube N., Josling G., Pasaje C.F., Kouskoumvekaki I., Panagiotou G., Niles J.C., Jacobs-Lorena M., Denise Okafor C., Gamo F.J, & Llinás M. (2022). Inhibitors of ApiAP2 protein DNA binding exhibit multistage activity against Plasmodium parasites. PLOS Pathogens, 18(10), e1010887.

Publication 2022

mouse anti-GFP ab38905 mouse anti-H3 goat anti-rat HRP conjugate goat anti-rabbit HRP conjugate autoradiography film

Aldolase Antibodies Antibody Autoradiography Bromophenol blue Buffer Goat Mouse Parasite Protein Rabbit Rbcs Saponin Tris Western blot

Corresponding Organization : Center for Disease Dynamics, Economics & Policy

Other organizations : Princeton University, Johns Hopkins University, Massachusetts Institute of Technology, Technical University of Denmark, Chinese University of Hong Kong, University of Hong Kong, Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e. V. - Hans-Knöll-Institut (HKI), GlaxoSmithKline (Spain)

Top 5 similar protocols

Variable analysis

independent variables

Parasite protein fractionation method described in [95]

dependent variables

Full parasite protein western blot samples

control variables

Lysis of RBCs with 0.1% saponin
Boiling of protein in Loading Buffer (50mM Tris-Cl pH 8.0, 20% SDS, 1% Bromophenol Blue)
Primary antibodies used: 1/1000 rat ant-HA (Roche 3F10), 1/1000 mouse anti-GFP (Roche), 1/3000 rabbit anti-aldolase conjugated to HRP (Abcam ab38905), or 1/3000 mouse anti-H3 (Abcam ab10799)
Secondary antibody concentrations used: 1/3000 goat anti-rat HRP conjugate (Millipore), 1/3000 goat anti-mouse HRP conjugate, or 1/10,000 (Pierce) goat anti-rabbit HRP conjugate (Millipore)
ECL reagent (Pierce) used to detect HRP signal
Blots exposed to autoradiography film (VWR) and visualized using an autoradiography developer

controls

Positive controls not explicitly mentioned
Negative controls not explicitly mentioned

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