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ALMUDENA CROOKE Y COLS. AN. R. ACAD. NAC. FARM.

For Western blot analyses, infected red blood cells (IRBCs) from a
10 ml-culture were suspended in 2 volumes of 0.2% saponin in PBS
and incubated for 20 min at 37º C, to lyse the RBC membranes. The
released parasites were pelleted at 10000 x g for 10 min and washed
three times in cold PBS. The parasite pellet was solubilized in 100 µl
of TTP (PBS containing 1% Triton X-100 and a protease inhibitor
cocktail, Complete Mini, Roche Diagnostics, Mannheim, Germany)
and incubated for 30 min at 4º C with frequent vortexing. Next,
the soluble parasite fraction was frozen (at –70º C for 5 min) and
thawed (at 37º C for 5 min) only once and centrifuged for 10 min at
10000 x g. 10 µg of total protein supernatant was boiled in 5x loading
buffer and separated on 10% SDS-PAGE. After electrophoresis,
proteins were transferred to PVDF membranes and probed with the
above mentioned antibodies at 1/200 dilutions. Antibody binding was
detected by incubation with secondary 1/5000 HRP-conjugated sheep
anti-rabbit Ig Abs, followed by chemoluminescence detection using
SuperSignal West Pico (Pierce Biotechnology Inc, Rockford, IL).
Bands were quantified using Quantity One® software (BioRad,
Hercules, CA).

RESULTS

PfG6PD-6PGL protein expression patterns across
the intraeryhrocytic cycle

Expression patterns at the protein level were examined through
immunodetection of parasite G6PD-6PGL using antibodies against
N- and C- terminal separately and a mixture of both of them. Bands
with different molecular weight were consistently observed in
western blots. As shown in Figure 2, a 107 kDa band corresponding
to the theoretical molecular mass of the deduced protein sequence
was abundant mainly in mature stages and detected with both
antibodies. Also two other abundant bands of 73 and 69 KDa were
observed, predominantly in the mature parasite. Thus, the 73 kDa
band, detected by the anti-6PGL domain antibody, has a size that
includes the N-terminal domain with the expected 6PGL activity and
the main low-complexity stretch between both domains. The 69 kDa
band, detected by the anti-G6PD domain antibody, match with the

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