Biomedical Engineering Reference
In-Depth Information
SERP, parasitophorous vacuolar space; PfAldolase, parasite
cytosol. In principle any antibodies that are verifi ed to be spe-
cifi c only to each of these cellular compartments may be used.
6. To do this as accurately as possible, cut the end off the end of
a 100
l pipette tip to create a larger opening.
7. For example, if you require 14
ʼ
ʼ
l of SLO, resuspend IE in
l PBS.
8. We fi nd that best results are achieved if the SLO solution is
pipetted onto the side of the reaction tube and then slowly
incorporated into the remaining cell suspension.
9. These centrifugation steps are designed to avoid unwanted car-
ryover of permeabilized cells in the supernatant fraction. For
added pipetting accuracy, put a 10
166
ʼ
ʼ
l pipette tip onto the end
l tip being used.
10. In our experience 5 × 10
6
cell equivalents is enough material to
obtain strong signals for the controls using the antibodies and
conditions suggested. Depending on the expression level of
the protein of interest, less or more equivalents may be loaded.
As noted above, in our experience loading more than 1 × 10
7
total cell equivalents (including non-infected cells) leads to
strong nonspecifi c signals during immunodetection. A higher
initial parasitemia (up to 95 % can be achieved using magnetic
cell sorting) allows more parasite cell equivalents to be loaded.
The formula takes into account the parasitemia, the initial
number of cells (2 × 10
8
), the required number of cells to be
loaded (5 × 10
6
), the volume of the sample (200
of the 100
ʼ
l) and for the
supernatant a factor (0.75) to correct for sample loss during
the procedure.
11. Tubes 1, 3, and 4 are controls: Tube 1, control that any prote-
olysis observed is only due to the addition of exogenous
Proteinase K and is not due to the activity of endogenous pro-
teases; tube 3, control that the protein of interest is Proteinase
K sensitive; tube 4, control that any proteolysis observed took
place prior to addition of sample buffer.
12. To divide SLO-permeabilized cells into equal amounts, calcu-
late the desired amount and split cells into four aliquots while
performing the third and last washing step. Then, centrifuge,
discard washing supernatant, and proceed according to
Table
2
. Start by resuspending the permeabilized cells in PBS.
ʼ
Acknowledgement
J.M.P. is supported by DFG Grant PR1099/3-1 as part of the
SPP1580. S.K. is supported by a postdoctoral DFG scholarship.
