• affinity labelling reagents are available from Bachem Bioscience, Calbiochem, Enzyme a limited number of suppliers, including Systems Products, and the Peptide Institute
1. Each sample should contain ~1 x 10® cells. At the very least, an experiment should contain cells treated with a pro-apoptotic stimulus and control (non-apoptotic cells).
2. After the induction of apoptosis is complete, wash cells twice in ice-cold serum-free isotonic buffer (e.g. PBS).
3. Isolate cytosol or other subcellular fractions using the techniques described In Protocol 2 and store at -70°C in buffer A supplemented with 5 mM EDTA.
4. Determine protein concentration in an aliquot of the subcellular fraction using an assay that is not affected by reagents in the EDTA-supplemented buffer A [e.g. the bichinconinic acid method <21)].
5. Perform affinity labelling by reacting 30-50 |xg of cytosolic protein for 1 h at 20-22°C with 1 n,M Z-EK(bio)D-aomk. The Z-EK(blo)D-aomk is conveniently prepared as a 25 mM stock in DMSO and stored in small aliquots at-80°C.
6. To stop the reaction, dilute the sample with 0.5 vol. 3 x concentrated SDS sample buffer [0.15 M Trls-HCI (pH 6.8 at 20°C), 45% (w/v) sucrose, 6 mM EDTA, 9% (w/v) SDS, 0.03% (w/v) bromophenol blue, 10% (v/v) p-mercaptoethanol] and heat to 95-100°C for 3 mln.
7. Subject the samples to one-dimensional SDS-PAGE In 16% (w/v) polyacrylamide gels. Alternatively, subject the samples to two-dimensional isoelectric focusing/SDS-PAGE. For the latter procedure, our collaborators have obtained highly reproducible results using pre-cast Immobiline gels (pH 4-7, Pharmacia, Upsalla, Sweden) for the first dimension.
8. After separation, transfer polypeptides to nitrocellulose.
9. Block unoccupied protein-binding sites with PBS containing 0.1% (v/v) Tween-20 (PBS-T) and 5% (w/v) non-fat dry milk.
10. Visualize the labelled polypeptides by reacting with peroxidase-coupled streptavldin in PBS-T for 3-4 h at 20-22°C. Wash blots with PBS-T for 1 x 15 min and 4x5 min to remove unbound peroxidase-coupled streptavidin.
11. Detect the bound peroxidase using enhanced chemilumlnescent detection.
12. Recombinant caspases expressed in Sf9 cells (18) can be subjected to this analysis in parallel in order to permit identification of the labelled polypeptide species. His6-tagged caspases (e.g. commercially available from Pharmlngen, San Diego, CA) should not be used as standards for two-dimensional analysis because the charged tag will alter their migration.
This assay can detect pg levels of active proteases. Moreover, when utilized in conjunction with known amounts of recombinant caspases, this assay can identify and quantify the individual proteases that are active at particular times during the course of programmed cell death.
Control experiments indicate that cytosol can be successfully labelled after storage at -70 °C for at least 3 months. As an alternative to subcellular fractionation, whole-cell lysates prepared by freezing and thawing cells can also be labelled using analogous techniques (43). The caspase labelling pattern in these lysates, however, is more complicated than the pattern observed using cytosol or nuclei, suggesting that specific caspase species might target other subcellular compartments as well.
The recommended 20-22 °C temperature for incubation with the affinity label appears to be important. Incubation of cytosol at 37 °C results in rapid degradation of active caspase species (P.W. Mesner, Jr, L. Miguel Martins, and S.H. Kaufmann, unpublished observations).
Two types of controls can be performed to distinguish endogenous biotinyl-ated polypeptides from species that become biotinylated as a consequence of reaction with Z-EK(bio)D-aomk. First, as indicated above, control (non-apoptotic) cells can be labelled in parallel with apoptotic cells. Although some non-apoptotic cells contain active caspase-1 (38), there is no evidence for active caspases-3, -6, or -7 in non-apoptotic cells. Secondly, the ability of other caspase inhibitors to prevent labelling can be assessed. For example, preincubation of cell lysates with certain peptide chloromethyl or fluoromethyl ketones will completely abolish subsequent labelling of active caspases with Z-EK(bio)D-aomk but will not have any effect on the reaction of streptavidin with endogenous biotinylated polypeptides (44).
It is also possible to combine affinity labelling with subsequent affinity chromatography on avidin-agarose to purify active caspases up to 100000fold in a single step (33, 38, 43,45).
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