Horseradish peroxidase hrp conjugated anti rabbit igg

ES, MS, and LS stage-specific cultures were harvested at 5% parasitemia, and parasite protein was isolated. Western blot analysis was performed as described earlier (21 (link)). The blot was probed with rabbit anti-TopoVIB antibody (5 (link)) and rabbit anti-human Spo11 (anti-hSpo11) antibody (Invitrogen) at 1:500 and 1:3,000 dilutions, respectively. In order to detect PfSpo11 or ScSpo11, we used anti-human Spo11 antibody. Mouse anti-human actin 1 antibody (Abcam) was used as a normalizing control. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Promega) and anti-mouse (Santa Cruz Biotechnology) were used as the secondary antibodies in a 1:10,000 dilution. To probe the protein level in the presence of atovaquone, cytochrome c (Cytc) was used as a mitochondrial protein marker. For that, atovaquone-treated parasite proteins were probed with anti-mouse anti-Cytc (Abcam) at 1:3,000 dilutions. The Western blots were developed by using a chemiluminescence detection system (Pierce). Each experiment was repeated with three independent batches of cells, and band intensities were quantified using ImageJ software. Mean relative densities were plotted using GraphPad prism.

Protein, 20–30 µg, was separated by electrophoresis on 10–12% SDS-polyacrylamide gels at 60–100 mV and then transferred onto polyvinylidene fluoride (PVDF) membranes for 90 min on constant 200 mA at 4 °C. After protein transfer, membranes were blocked in 5% BSA + Tris-buffered saline plus Tween-20 (TBST) blocking buffer for an hour, then incubated with the respective primary antibodies overnight at 4 °C. Following 3 washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Promega,#W401B, Madison, WI, USA) or HRP-conjugated anti-mouse IgG secondary antibodies (BioRad, #170-6516, Mississauga, ON, Canada) (1:5000) for 1 h at room temperature. Signals were detected using ECL plus kit reagents (Perkin Elmer, Guelph, ON, Canada) on a Chem Doc imager (BioRad, Mississauga, ON, Canada). Primary antibodies used were: Anti-Caspase-12 (Abcam, #ab62484, Toronto, ON, Canada); Anti-KDEL (Abcam, #ab176333, Toronto, ON, Canada), which recognizes GRP94, GRP78, and PDI; Anti-XBP1(Abcam, #ab37152, Toronto, ON, Canada); Anti-ATF6 (Proteintech, #24169-1-AP, Rosemont, IL, USA); Anti-PERK (Proteintech, #20582-1-AP, Rosemont, IL, USA); Anti-IRE1α (Proteintech, #27582-1-AP, Rosemont, IL, USA).

Each sample (2 μl) was applied to a nitrocellulose membrane, blocked with 10% nonfat milk in Tris-buffered saline with Tween (TBS-T) overnight at 4°C, washed three times for 5 min each with TBS-T, and incubated for 1 h at RT with the anti-APF antibody αAPF (1:1000) for the synthetic samples and the anti-tau antibody Tau-5 (1:5000) for the immunoprecipitated samples. The membranes were washed three times for 5 min each with TBS-T, incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Promega, Madison, WI) diluted 1:10000 in 3% BSA/TBS-T, and incubated for 1 h at room temperature. The blots were washed 3 times with TBS-T and developed with an enhanced chemiluminescence kit from (RPN2132; Amersham-Pharmacia/GE Healthcare Life Sciences, Little Chalfont, UK).