One possible reason may be that the cells that you are using are responding to etoposide by DNA damage-induced necrosis rather than apoptosis. Positive PI staining is not a specific test for apoptosis (it is positive for late apoptosis but also for necrosis). You may want to try either a different apoptosis-inducing agent (e.g. staurosporine) or a different cell line. It is also recommended to do dose-response curve or to monitor the marker of apoptosis at different time-points. Cleaved caspase-3 can peak at different times depending on cells, inducing agents and their concentration (in HepG2 cells and tamoxifen it can be 1-24 hrs depending on concentration). Anyway, in this case I would rather go with more high-throughput experiment than WB, such as quantification of caspase-3 activity by fluorescent or luminescent assay in a 96-well plate (more convenient way to determine optimal conditions in many replicates at the same time, more reliable quantification...).

CC3 can be tricky to detect...I always load AT LEAST 50 micrograms of lysate to detect CC3.  I also use a cell signaling antibody but a different catalog number.  I have used the 9661 at 1:1000 overnight and always had great results.  I use the same transfer conditions and PVDF membranes so I don't think that is a problem. I would also suggest not running a 15% gel; try a 12% gel.  I have had problems with CC3 detection on a 15% gel.  I don't know why this is, but a 12% should work.

Good Luck!

You have mentioned about actin in your question. But it is not clear that whether you can see it or not in your WB. If you can see your actin band than there may be some problem with your antibody. But i will suggest you to repeat the experiment including some other cell line. and if you have Staurosporin in your lab you can use it as your additional control. 

Since you can detect procaspase and not cleaved caspase-3, you might be running the gel or tranferring for too long (maybe loosing the low molecular weight proteins). Do you usually check if you have low molecular weight proteins in you membrane after transferring by staining it with Ponceu? If they are in the membrane, you may have a weak signal for the cleaved caspase-3, so you should exposure a little longer to detect it. 

I was able detect cleaved caspase-3 by WB using a gradient gel of 5-20% SDS-PAGE, running 30 micrograms of protein and transferring to nitrocellulose membrane. Primary antibody incubation was performed overnight.

Good luck!

One possible reason may be that the cells that you are using are responding to etoposide by DNA damage-induced necrosis rather than apoptosis. Positive PI staining is not a specific test for apoptosis (it is positive for late apoptosis but also for necrosis). You may want to try either a different apoptosis-inducing agent (e.g. staurosporine) or a different cell line. It is also recommended to do dose-response curve or to monitor the marker of apoptosis at different time-points. Cleaved caspase-3 can peak at different times depending on cells, inducing agents and their concentration (in HepG2 cells and tamoxifen it can be 1-24 hrs depending on concentration). Anyway, in this case I would rather go with more high-throughput experiment than WB, such as quantification of caspase-3 activity by fluorescent or luminescent assay in a 96-well plate (more convenient way to determine optimal conditions in many replicates at the same time, more reliable quantification...).

what antibody are you using to detect caspase-3?  Is it possible that it is not recognizing the cleaved form (i.e. the epitope bound by the antibody is either eliminated by cleavage, or present only as a small fragment that either runs off the bottom of your gel or blows through your membrane during transfer)?

only PI staining is not sufficient to measure apoptosis. Try TUNEL or AnnxV. cleaved caspase 3 is not very stable. after activation of caspase 3 it was consumed by the substrate. my suggestion is that try a lower time point or lower does treatment by etoposide. do you get reduction of total caspase 3? if so my feeling is due to the extensive treatment, all the activated form of caspase 3 was consumed and you did not identified a CC3 band. It also depends the type of the cell on which you are treating your drug. Does upstream caspases like C8/C9 activated in your treatment? this also give you information that CC3 formation is expected or not.

All the best for your future experiment.

Assessment of activity of cleaved caspase-3 by using tetrapeptide substrate is the ideal method than western blot because some times the low molecular weight proteins may run out of the gel or sometimes it is very difficult to find out the  time where the exact mechanism is happened. sometimes we may lyse the cells before or after the cleavage, then we cannot able to see in western blot.

thank you

But do you see disappearance of the pro-caspase? Otherwise, as mentioned, you may be killing cells by necrosis.

Do you have a positive ctrl such as STS or TRAIL?

Are you sure your antibody detects cleaved C3?

Once you have checked all the suggestions above, if you still can't see it you may also consider that it may be caspase-independent apoptosis, I saw something similar in Ovarian cancer cells...that's years ago, I could se mitochondrial depolarisation, activation of C9, C8 but not C3. You may want to check those as well.

Hi,

Cleaved fragments (activated caspases) are very unstable and are degraded further in the cell. It is very important to vary your time point of treatment. you may for example observe cleaved fragment of Mol wt 43 at 12 hr, 23 at 18 hr but none of them at 24 hr of treatment. You just do the time kinetics and you may also use the lower dose of the drug/or ........ for the same.

Hope you will get your results soon and you can check caspase-2 blot in the paper attached.

Timing, protease inhibitors, loading more protein, longer transfer times, smaller PVDF poor size and Femto Chemi. Those are all the tricks I could use to get a decent signal. 

Here trying to help and someone just gave a negative score to my previous answer....I would like to understand why.

You can try Cleaved PARP which is more broader apoptotic marker than caspase 3. You can also try immunocytochemsitry for cleaved caspase 3 on cells seeded on coverslips.

Adding ZVAD to the lysis buffer in addition to a good protease inhibitor cocktail seems to help stabilize the cleaved fragments, as well as preventing any cleavage after the cells are lysed.  I would also be careful not to transfer too long since small fragments can actually go through the membrane.  Fixing the membrane with methanol immediately after the transfer will help to more firmly bind the small fragments to the membrane, simply dip the PVDF membrane into 100% methanol for 10 seconds, place protein side up on a paper towel to dry, return the dry membrane to the methanol for another 10 seconds and then was with water and your normal wash buffer and block.  This method will also allow the blot to be stripped more times since all the proteins will bind more tightly.

also try with more ug of protein and also cut the membrane in the MW of cleaved caspase 3