Biomedical Engineering Reference
In-Depth Information
6. Add the amount of activated SLO solution noted in the table
below to each tube. Mix gently to resuspend and incubate for
6 min at RT. During this time, invert the tubes ten times every
2 min.
7. Isolate supernatant by centrifugation at 1,000 ×
g
for 4 min.
Remove 180
l of each supernatant to new tubes.
8. Generate a 100 % positive lysis control. Add 20
ʼ
ʼ
l of RBC to
180
l PBS. Lyse by three freeze/thaw cycles. Centrifuge as
above and remove 180
ʼ
ʼ
l supernatant to a new tube.
9. Make 800
ʼ
l of a 1/200 dilution of each supernatant (4
ʼ
l in
l of each dilution, in triplicates, into
a microtiter plate suitable for the plate reader available.
10. Measure absorbance at 412 nm. This gives a measure of the
amount of hemoglobin released.
11. Graph amount of hemoglobin released against volume of SLO
used. Set the positive control to 100 %. 1 hemolytic unit (HU)
is the amount of SLO required to cause 50 % cell lysis.
796
ʼ
l PBS). Aliquot 200
ʼ
1. Wash infected erythrocytes (IE) and resuspend 2 × 10
8
purifi ed
infected erythrocytes in 200
3.2 Permeabilization
and Subcellular
Fractionation of
Infected Erythrocytes
ʼ
l PBS. Centrifuge at 1,000 ×
g
for
2 min at RT.
2. Resuspend IE in a volume of PBS equivalent to (180
ʼ
l −
(volume of SLO required to have 4 HU)) (
see
Note 7
).
3. Activate SLO. Remove required number of aliquots of SLO
from storage and thaw. To each add 9
ʼ
l of 1 M DTT. Mix
gently and incubate at RT for 15 min.
4. Add required amount of SLO solution to each tube. Mix gen-
tly to resuspend and incubate for 6 min at RT (
see
Note 8
).
During this time, invert the tubes ten times every 2 min.
5. Centrifuge samples at 1,000 ×
g
for 4 min at RT. From each
sample, remove 180
l of supernatant (SNT) to a new tube.
Retain pellet (P). Re-centrifuge SNT at 1,000 ×
g
for 4 min
at room temperature. Remove 150
ʼ
ʼ
l of SNT to a new tube
(
see
Note 9
).
6. Wash P three times in 500
ʼ
l PBS (1,000 ×
g
, 4 min, RT).
7. Add 50
l of 4× protein sample buffer to each supernatant.
Resuspend each pellet in 150
ʼ
ʼ
l of PBS then add 50
ʼ
l of 4×
protein sample buffer to each. Boil samples for 10 min.
8. Calculate the volume of sample (in
l) required to load 5 × 10
6
infected cell equivalents of both SNT and P using the follow-
ing formulae (
see
Note 10
):
Supernatant
:
ʼ
Volume
to
load =
(5 × 10
6
÷ ((2 × 10
8
×
(% parasitemia ÷ 100) × 0.75) ÷ 200)).
Pellet
: Volume to load = (5 × 10
6
÷ ((2 × 10
8
× (% parasitemia ÷
100)) ÷ 200)).
