Activation of apoptotic activity in cytoplasmic extracts and measurement of the resulting caspase enzymatic acticity
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Activation of apoptotic activity in cytoplasmic extracts for western blot analysis
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Reconstitution of activated cytoplasmic extracts with isolated nuclei. Analysis of apoptotic activity by qualitative DNA laddering assay and DAPI staining.
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Reconstitution of activated cytoplasmic extracts with isolated radioactive nuclei. Analysis of apoptotic activity by quantitative, radioactive DNA fragmentation assays or DAPI staining.
Radioactive DNA fragmentation assay Radioactive nuclei (e.g from ALVA31 cells) were prepared as described in the protocol "Isolation of cell nuclei for the application in a cell-free system". Prior to use in the cell-free system, 5x104 nuclei in NSB (5 µl of 107 nuclei per ml) were distributed in 0.5 ml microfuge tubes and were washed once in 50 µl DB. The nuclei were then incubated in cytoplasmic extracts (final protein concentration 7.5 mg/ml) in the presence or absence of 10 µM cyt c and 1 mM dATP in a total volume of 10 µl for 4 h at 37°C (650 nuclei/µg protein). Here is an example for a reaction mix for this kind of experiment:
y corresponds to 75 µg protein, and x is calculated so that the final volume is 10 µl. After incubation, the nuclei were transferred from the microfuge tubes into the wells of a 96 well plate; the nuclei's DNA was harvested on a glassfiber membrane and the retented radioactivity measured by scintillation counting. Experiments were run in triplicate or pentuplicate for each condition. The percentage of DNA fragmentation was calculated as follows: ( [cpm of nuclei in pure extracts] - [cpm of nuclei in extracts + cyto c/dATP] ) / [cpm of nuclei in pure extracts] x 100
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MATERIAL |
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Extract Dilution Buffer (DB): |
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COMPOSITION: |
RECIPE for 500 µl: |
10 mM HEPES (pH 7.0) supplemented with ATP regeneration system:
2 mM ATP |
100 µl of 50 mM HEPES, pH 7.0 incl. -
5 µl of 200 mM ATP in water 365 µl H2O nuclease free |
Add DTT and ATP regeneration system always fresh to the buffer, just before use!
Caspase Assay Buffer (CAB): |
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COMPOSITION: |
RECIPE for 50 ml: |
50 mM PIPES |
838.4 mg PIPES
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add 40 ml H2O, adjust pH=7.2 using KOH (about 200 µl of 1M KOH), then fill up to the final volume of 50 ml.
1mM DTT is always added fresh to the buffer, just before use.
Lysis Buffer (for DNA isolation from nuclei) |
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COMPOSITION: |
RECIPE for 50 ml: |
50 mM Tris-HCl, pH 8.0 |
5 ml of 0.5 M Tris-HCl, pH 8.2 add 43 ml H2O
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TE Buffer |
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COMPOSITION: |
RECIPE for 50 ml: |
50 mM Tris-HCl, pH 8.0 |
5 ml of 0.5 M Tris-HCl, pH 8.2 add 44.9 ml H2O
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4% Paraformaldehyde in PBS |
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RECIPE for 100 ml: |
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Solution is good for at least 1 year. |