166
- Place the cassette into electrode assembly and fi ll the tank with
cold transfer buffer. - Connect the electrophoresis unit to the power supply and
make sure that the direction of transfer is correct. - Perform the transfer at a constant 100 V for 2 h (alternatively,
120 V for 1.5 h) at 4 °C. Transfer effi ciency is indicated by
transfer of prestained SDS-PAGE markers ( see Note 23 ). - Transfer the membrane into blocking solution. Incubate at
least 1 h on a rocker. - Rinse the membrane several times in PBS.
- Put the membrane into a small plastic container ( see Note 24 ).
Add the diluted primary antibody of interest (usually from
1:500 to 1:5,000 in diluent solution). Incubate overnight on a
rocker at 4 °C ( see Note 25 ). - Wash three times (10 min in PBS, 15 min in PBS-T, 10 min in
PBS) on a rocker. - Add peroxidase-conjugated secondary antibody diluted in
blocking buffer (usually 1:5,000 to 1:10,000). Incubate for
1 h on a rocker. - Wash as in step 4.
- Develop membrane with ECL according to the manufacturer’s
instructions. - Remove excess PBS and wrap the membrane in plastic foil.
Expose the membrane to X-ray fi lm in a developing cassette.
Bands corresponding to zymogen and processed caspases are
normally detected within 1–10 min of exposure ( see Notes 26
and 27 ). - Process the fi lm in an automatic X-ray developer.
- Before reprobing the membrane using other primary antibodies,
incubate the membrane in stripping buffer for 30 min at 50 °C
with agitation ( see Notes 28 – 30 ). - Wash the membrane extensively with water and repeat steps 1 – 9.
- Place coverslips in 6-well or 12-well culture plates.
- Seed cells and allow them to attach to the glass surface ( see
Notes 31 and 32 ). - Induce apoptosis in cells by desired way. Concurrently incu-
bate a control culture without induction. - Aspirate cell culture medium and rinse cells twice in cold PBS.
- After the last wash, cover cells with 4 % cold formaldehyde and
incubate the samples at 4 °C for 15–30 min. - Wash the fi xed cells 3× 5 min in PBS ( see Note 33 ).
3.1.4 Immunodetection
3.2 Immuno-
cytochemical
Detection of Active
Caspases
3.2.1 Sample
Preparation
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