Western Blot Analysis
Asexual blood stage parasites of WT NF54, lines MDV1- and
G377-TurboID-GFP, line PPLP2-GFP-BirA and the HA-glmS -tagged
parasite lines were harvested from mixed cultures. For erythrocyte
lysis, parasites were incubated for 10 min in 0.05% (w/v) saponin/PBS.
Gametocytes were enriched by Percoll purification as described above.
Pelleted parasites were resuspended in lysis buffer (150 mM NaCl, 0.1%
(v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 50
mM Tris-HCl, pH 8.0) supplemented with protease inhibitor cocktail (PIC,
Roche; Basel, CH). A 5x SDS loading buffer with 25 mM dithiothreitol was
added to the lysates, followed by heat-denaturation for 10 min at 95°C.
Lysates were separated via SDS-PAGE and transferred to a nitrocellulose
membrane. Non-specific binding sites were blocked by incubation with 5%
(w/v) skim milk and 1% (w/v) BSA in Tris-buffered saline (pH 7.5) for 1
h at 4°C. For immunodetection, membranes were incubated overnight at 4°C
with monoclonal mouse anti-GFP antibody (Roche; Basel, CH; dilution
1:500) or monoclonal rat anti-HA antibody (Roche; Basel, CH; dilution
1:200) and polyclonal rabbit anti-Pf39 antisera (dilution 1:10,000) in
3% (w/v) skim milk/TBS. The membranes were washed 3x each with 3%
(w/v) skim milk/TBS and 3% (w/v) skim milk/0.1 % (v/v) Tween/TBS and
then incubated for 1 h at RT with goat anti-mouse, anti-rabbit or
anti-rat alkaline phosphatase-conjugated secondary antibodies (dilution
1:10,000, Sigma-Aldrich; Taufkirchen, DE) in 3% (w/v) skim milk/TBS.
The membranes were developed in a NBT/BCIP solution (nitroblue
tetrazolium chloride/5-bromo-4-chloro-3-indoxyl phosphate; Roche; Basel,
CH) for up to 20 min at RT. For the detection of biotinylated proteins,
the blocking step was performed overnight at 4°C in 5% (w/v) skim
milk/TBS and the membrane was washed 5x with 1x TBS before incubation
with streptavidin-conjugated alkaline phosphatase (dilution 1:1,000,
Sigma-Aldrich; Taufkirchen, DE) in 5% (w/v) BSA/TBS for 1 h at RT.