171
(c) Remove stain by repeated washing in water or PBS.
(d) Proceed to the blocking procedure.
- To minimize evaporation and the required volume of primary
antibody solution, choose a lidded container with a size similar
to that of the membrane.
- For high abundant protein targets, incubation for 1 h at room
temperature is suffi cient.
- A number of factors, including quality of antibodies and the
type of ECL used, may infl uence exposure times considerably.
- In some experimental models, processed caspase fragments
may be hard to reveal. This is, for some reason, especially true
for caspase-2. One approach to overcome this problem is to
fi rstly perform immunoprecipitation using agarose-conjugated
VAD (agarose-valyl-alanyl-aspartyl), which bind the catalytic
site of active caspases. Subsequently, precipitated material is
separated in SDS-PAGE and immunoblotted with the anti-
body of interest [ 16 ].
- By repeating the immunodetection step, it is possible to target
other caspases in the same membrane Stripping is only required
if the proteins targeted are overlapping in size or can be recog-
nized by the same secondary Abs. If this is not the case, wash
the membrane 3 × 10 min in PBS and proceed to immunode-
tection ( steps 1 – 9 ) using a second primary antibody of
interest.
- Markers such as G3PDH or b -actin, which expression levels
remain relatively constant across various cell culture condi-
tions, should also be detected. Optical density values of target
proteins bands are then correlated to the corresponding values
of the marker protein. By this mean, comparisons in between
individual samples become more accurate.
- Long-term storage of nitrocellulose membranes is accomplished
by drying. During storage, keep the membranes protected in
between two 3MM papers. For repeated use, incubate the
membranes in PBS for 30 min on a rocker and proceed to
immunodetection.
- For some cell types poly- L -lysine-coated coverslips may be
required as culture substratum.
- For cells growing in suspension, a cytospin protocol is required
in order to attach cells to the coverslip.
- Fixed and washed samples can be stored at 4 °C for up to 1 week.
- When a primary antibody is used in immunocytochemistry for
the fi rst time, it should be titrated to determine the dilution
which allows for a robust specifi c signal with a minimum of
background (normally 1:200 to 1:1,000).
- The surface tension will keep the liquid in place.
Caspases in Mammalian Cell Cultures
