The protocol is performed after drug treatment on the cancer cell lines. Please provide necessary details and intricacies, as I am new to this technique.
For apoptosis, caspases are normally assayed with kits. The kits can use colorimetric or fluorescent readouts. I would recommend do this as well. This type of asay takes just a few minutes.
The proteins to look at in the western would be some of the caspase members, like 3. Maybe others can comment on which proteins and Abs to use.
Another comment, I would use complete protease inhibitor cocktail pellets if you do the lysis as described above. I personally would lyse the cells in buffer with SDS too.
I find it very useful to add ZVAD, a pancaspase inhibitor, to the lysis buffer along with the usual protease inhibitors. This gives much better results when trying to compare the degree of caspase cleavage in different treatments, by preventing additional cleavage after lysis. Some other related proteins such as cFLIP can also be cleaved by caspases. I have done quite a bit of work with cFLIP and found that the addition of ZVAD to the lysis buffer was the only way to get reproducible results in western blots. Without the ZVAD the cFLIP levels would decrease quickly if the gel wasn't run immediately after preparing the lysates. I personally prefer the Pierce protease inhibitor cocktail, it is already solubilized in DMSO and can be purchased pre aliquoted for storage at 4C; this is much more convenient the working with individual (and often toxic) protease inhibitors and there is no problem with solubility (which can sometimes occur with the tablets). Cell Signaling has some very good antibodies for cleaved caspase-8 which are especially helpful if you are studying the extrinsic pathway. If you decide to use any type of kit to assay for caspase activity as mentioned above, make sure it includes a proteasome inhibitor to block the caspase-like activity of the proteasome; the Promega caspase-glo kits have worked well for me, but they are expensive. Cell Signaling Technologies also sells an apoptosis antibody kit, with smaller amounts of their most popular antibodies. These may be a good set to start with. To save money make up the primary antibodies in a BSA based buffer (rather than milk) and add a preservative (such as sodium azide), then the diluted antibody can be stored at 4C and reused for multiple blots. Just be sure to wash any milk off your blot before adding then antibody.
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