2.2. Immuno-affinity capture and in vivo crosslinking

Immuno-affinity capture of GFP fusion proteins was carried out by GFP pull-down using the μMACS GFP tagged protein isolation kit (Miltenyi Biotec) according to manufacturer's instructions. Briefly, 5–10 million purified parasites were lysed in 1 ml of pre-cooled lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl pH 8.0) and incubated on ice for 30 min. Cell debris was removed by centrifugation at 10,000 ×g. Fifty μl anti-GFP microbeads were added to the supernatant and the mixture incubated on ice for 30 min to allow antibody binding. The cell lysate was then run by gravity through a magnetic microcolumn to capture the magnetic microbeads, followed by four 200 μl washes with lysis buffer. Proteins were eluted in 50 μl pre-heated 95 °C elution buffer (50 mM Tris-HCl pH 6.8, 50 mM DTT, 1% SDS, 1 mM EDTA, 0.005% bromophenol blue, 10% glycerol) and frozen until further use. For in vivo crosslinking, purified ookinetes were collected by low speed centrifugation (0.8 xg), resuspended in 0.5 ml PBS supplemented with 1% (w/v) paraformaldehyde and incubated at room temperature. The cells were collected by centrifugation after a total of 10 min in the fixative (including centrifugation), resuspended in 0.5 ml 250 mM Tris-HCl (pH 7.2), and incubated 10 min at room temperature to quench the formaldehyde. Cells were again collected by centrifugation followed by cell lysis and GFP pull-down as described.