Western breeze chemiluminescent anti rabbit kit

Activated gametocyte and ookinete samples were isolated as described below. WT or SAS6L-GFP samples were then purified using a GFP-Trap kit to immunoprecipitate GFP-fusion protein (Chromotek). After the addition of Laemmli sample buffer, the samples were boiled and loaded on a 4–12% SDS-polyacrylamide gel. Samples were subsequently transferred to nitrocellulose membranes (Amersham Biosciences) with immunoblotting performed using the Western Breeze Chemiluminescent Anti-Rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a concentration of 1:1250, according to the manufacturer’s instructions.

Parasite-infected red blood cells were placed in schizont culture medium (RPMI 1640, FCS 1:10, Penicillin/Streptomycin 1:100) for 24 h at 37°C and parasites were allowed to mature to schizonts, which were purified the following day on a 60% v/v NycoDenz (in PBS) gradient, harvested from the interface and washed (stock solution: 27.6% w/v NycoDenz in 5 mM Tris-HCl, pH 7.20, 3 mM KCl, 0.3 mM EDTA). After the addition of Laemmli sample buffer to the cells, the samples were boiled and electrophoresed on a 4–12% SDS-polyacrylamide gel. Resolved proteins were subsequently transferred to nitrocellulose membranes (Amersham Biosciences) and immunoblotting performed using the Western Breeze Chemiluminescent Anti-Rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a concentration of 1:1250, according to the manufacturer's instructions.
A schizont lysate was also used for immunoprecipitation with a GFP-Trap^®_A Kit (Chromotek) following the manufacturer's instructions. The GFP-Trap^®_A beads were equilibrated with dilution buffer and proteins bound from the parasite lysate by slow mixing at 4°C for 2 h. The beads were then harvested by centrifugation at 1200 g for 2 min and washed twice with dilution buffer. The beads with bound proteins were then treated with trypsin and released peptides were analyzed by tandem mass spectrometry.

The protein extractions and Western blotting were performed as described by Jelsbak et al.^{40 (link)}. The membranes were pre-blocked with human IgG to avoid a signal from ProteinA. The rabbit-raised antibodies against staphylococcal Sle1^{48 (link)}, Spx^{49 (link)} and ClpX^{40 (link)}. The bound antibody was detected using the WesternBreeze Chemiluminescent Anti-Rabbit kit or Anti-mouse kit (Invitrogen). All of the Western blots were repeated at least three times with similar results.

Cells for isolation of total proteins were harvested at the same time-points and from the same cultures as that used for RNA preparation (Northern blot analysis described above). One milliliter aliquots were harvested and kept at −80 °C, until all samples were collected. The cell pellets were thawed on ice, suspended in 50 mM TrisHCl, pH = 7.4, to a calculated OD₆₀₀ of 10.0. PMSF, Dnase, Rnase, and lysostaphin were added to the samples, and they were incubated at room temperature for 15 min. Cellular debris was removed by centrifugation and the protein concentration of each sample was measured using the Bradford dye-binding procedure from Bio-Rad. For immunoblotting, a total of 5 μg of each sample was loaded on NuPAGE^® 4–12% Bis-Tris gels (Invitrogen), using MOPS-Buffer (Invitrogen). The proteins were blotted onto a polyvinylidene difluoride membrane, using the XCell II blotting module (Invitrogen). The membranes were pre-blocked with IgG, before probing with antibodies directed against the Rot transcriptional regulator (Rabbit-anti-Rot-antibody [37 (link)]) at a 1:2000 dilution. Bound antibody was detected with the WesternBreeze Chemiluminescent Anti-Rabbit kit or Anti-mouse kit (Invitrogen). The Western blot analysis was repeated three times with similar results.

Schizont, gametocyte and ookinete samples were isolated as described below. WT-GFP or CYC3-GFP samples were then purified using a GFP-Trap kit to immunoprecipitate GFP-fusion protein (Chromotek). After the addition of Laemmli sample buffer, the samples were boiled and an equal concentration of total protein was loaded on a 4–12% SDS-polyacrylamide gel. Samples were subsequently transferred to nitrocellulose membranes (Amersham Biosciences) and immunoblotting performed using the Western Breeze Chemiluminescent Anti-Rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a concentration of 1:1250, according to the manufacturer's instructions.