Biomedical Engineering Reference
In-Depth Information
9. Load 5 × 10
6
cell equivalents on a 12 % SDS-PAGE using
standard protocols and including commercial protein stan-
dards. Load supernatant and pellet fractions from each sample
next to each other on the gel to allow direct comparison of
band intensity. One entire set of samples is required for detec-
tion of control proteins.
10. Transfer to nitrocellulose using standard Western blotting
protocols.
11. If using non-stained protein marker, confi rm correct transfer
by Ponceau S staining the membrane and mark marker bands
with pencil. Destain membrane with PBS.
12. Carry out immunodetection. If using antibodies with known
specifi city (such as those suggested) all control proteins can be
detected by carefully cutting the control membrane into three
sections as shown in Fig.
2a
. A correct result after permeabili-
zation is shown diagrammatically in Fig.
2b
.
3.3 Protease
Protection Assay
to Determine Protein
Topology
1. Label four tubes (1-4) (
see
Note 11
).
2. Use an equivalent of 5 × 10
7
infected SLO-permeabilized cells
(washed three times, from above protocol
step 6
) per tube
(
see
Note 12
).
3. Referring to Table
2
, add the required amounts of PBS,
Proteinase K (PK), Triton X-100 (T-X-100), PMSF, and PIC.
4. Mix gently and place samples on ice for 30 min.
5. Add PMSF to a fi nal concentration of 2 mM and PIC to 1× to
all samples to inactivate PK.
6. Add 16.5
l of 4× protein sample buffer to each sample and
boil for 10 min.
7. Load 5 × 10
6
cell equivalents per lane on a 12 % SDS-PAGE
using standard protocols and including commercial protein
standards.
8. Transfer to nitrocellulose membrane using standard Western
blotting protocols.
9. If using a non-stained protein marker, confi rm correct transfer
by Ponceau S staining the membrane and mark marker bands
with pencil. Destain membrane with PBS.
10. Carry out immunodetection. As a control for the effectiveness
of PK treatment, detect PfSBP1 or PfExp1 using the antisera
listed in Subheading
2
. Figure
2c, d
shows a diagrammatic rep-
resentation of a correct result.
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