Biomedical Engineering Reference
In-Depth Information
4
Notes
1. The original published protocol called for a cysteine-alanine
mutant of SLO that is constitutively active (C530A [
16
]).
However this mutant is not commercially available and thus
this current protocol describes use of only the commercially
available SLO variant which requires activation with DTT prior
to use. Those wishing to use the cysteine-alanine mutant are
requested to contact Professor Sucharit Bhakdi (University of
Mainz, sbhakdi@uni-mainz.de) for further information.
2. SLO does not remain stable at 4 °C for extended periods of
time. Be aware that, even when stored correctly SLO loses
activity over time, and may have to be re-assayed to ensure the
correct number of hemolytic units is used.
3. Parasites may be purifi ed from cultures using various pub-
lished methods including gelatine fl otation, percoll gradient
or magnetic activated cell sorting [
17
-
20
]. Parasitemia of at
least 50 % is required to ensure that it is possible to load
enough parasite-derived material on SDS-PAGE gels without
problems due to the high hemoglobin content. In our experi-
ence, no more than 1 × 10
7
hemoglobin-containing cell equiv-
alents can be loaded on a typical SDS-PAGE gel without
considerable nonspecifi c reactivity of hemoglobin with the
ECL reagent used during immunodetection. The resulting
high background typically obscures signals of interest. SLO
lysis is most successful on parasites at approx. 24 h post-
invasion. At later time points we note that the PVM appears to
become more fragile, and release of PV contents upon SLO
treatment occurs. For this reason, all cultures should be syn-
chronized to ensure a maximal 4 h spread of parasite develop-
ment, otherwise the presence of later stage parasites may lead
to leakage of control proteins into inappropriate fractions. For
reasons that are not clear, this protocol is not scalable. If more
than 2 × 10
8
cells per sample are to be permeabilized, they
must be split into aliquots of 2 × 10
8
for permeabilization to
be effective. Resulting subcellular fractions may be pooled fol-
lowing permeabilization.
4. While establishing this protocol we were surprised to note that
exogenously added proteinase K, and also endogenous cellular
proteases retain a certain level of activity in reducing SDS-
sample buffer. It is thus important to inactivate all proteases
before adding SDS-sample buffer. Any commercially available
PIC is suitable but should be supplemented by 2 mM PMSF.
5. We describe here the antibodies used in our laboratory as they
have been verifi ed to be specifi c to the following cellular com-
partments: HsHsp70, cytosol of the erythrocyte; PfSERA5/
