Western breeze chemiluminescence anti rabbit kit

Immunoprecipitation and subcellular fractionation of lysates containing GFP-tagged DEH was performed as described previously (Guttery et al, 2014 (link)). WT-GFP was used as the control protein in all experiments. In summary, cells from mouse blood infected with the DEH-GFP–expressing parasite were pelleted and then lysed in hypotonic buffer (10 mM Tris–HCl, pH 8.4, 5 mM EDTA) containing protease inhibitors (Roche, Cat. no. 04693159001), freeze/thawed twice, incubated for 1 h at 4°C, and then centrifuged at 100,000g for 30 min. The supernatant was collected as the soluble protein fraction (cytosol). The pellet was resuspended and washed in carbonate solution (0.1M Na₂CO₃, pH 11.0) containing protease inhibitors (Roche, Cat. no. 04693159001), and after incubation for 30 min at 4°C, the sample was centrifuged again at 100,000g for 30 min. The supernatant was saved as the peripheral membrane protein fraction (PMF). The residual pellet was solubilized in 4% SDS and 0.5% Triton X-100 in PBS, to form the integral membrane protein fraction (IMF). Samples from these three fractions, containing equal amounts of protein, were then analyzed by Western blot using anti-GFP polyclonal rabbit antibody (Invitrogen, Cat. no. A11122) and the Western Breeze Chemiluminescence Anti-Rabbit kit (Invitrogen, Cat. no. WB7106).

For western blotting, purified schizonts were lysed using lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% NP-40 and 1% sarkosyl). The samples were boiled for 10 min after adding Laemmli sample buffer to the lysed cells. The sample was centrifuged at 13,500 g for 5 min and electrophoresed on a 4–12% SDS–polyacrylamide gel. Subsequently, resolved proteins were transferred to nitrocellulose membrane (Amersham Biosciences) and immunoblotting was performed using the Western Breeze Chemiluminescence anti-rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a dilution of 1:1250, according to the manufacturer's instructions.