Murine anti ha antibody

The bacteria were harvested 2 h after induction and washed twice with ice-cold Tris-buffered sucrose (pH 7.0, 10 mM MgCl₂, 250 mM sucrose). After centrifugation, the bacteria were disrupted by glass beads (106 micron, Sigma) using a FastPrep^®-24 instrument (MP Biomedicals) to obtain protein extracts. After cell disruption and centrifugation, cell-free supernatants were mixed with SDS-PAGE sample buffer. After boiling the samples for 7 min, they were applied to 10% Stain-Free gels (Biorad), followed by electrophoresis with subsequent immunoblotting using the iBlot system (Thermo Fisher Scientific) according to the instructions of the manufacturer. Protein detection was performed with the SNAP i.d. System (Millipore), using a murine anti-HA antibody (Sigma) and a horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (BioRad). Proteins were visualized using the SuperSignal West Pico chemiluminescent substrate (Pierce).

For the detection of recombinant proteins in transgenic parasite lines, whole parasite protein extracts were prepared from saponin-lysed IRBCs as described in Methods in Malaria Research [64 ]. Proteins were loaded on standard discontinuous SDS-polyacrylamide gels and transferred to Hybond C membranes (Amersham). After blocking with 4% skimmed milk in 1xPBS/0.1% Tween20, HA-tagged proteins were recognized using a murine antiHA antibody (Sigma-Aldrich) and afterwards an antiMouse IgG-peroxydase antibody (KPL). Blots were exhaustively washed with PBS/Tween between incubations and finally incubated with WesternPico Super signal substrate (Pierce/Thermo). As a loading control, a murine antiPTEX150 antibody was used. Chemoluminescent signals were captured in an ImageQuant (GE) apparatus and intensities were quantified using ImageJ software (NIH). The obtained values were normalized using the PTEX150 signals.