Caspases are a group of cysteine proteases that cleave proteins after aspartic acid residues. To date, there are 14 known caspases that are divided into two main classes: initiators (or activators) and executioners (or effectors). Caspases exist as immature pro-caspases that must first be cleaved in order to become active. It has long been understood that caspases are essential proteins for the induction of apoptosis (programmed cell death).
Caspase-3 is considered the primary executioner caspase because it cleaves other important proteins that are necessary to induce apoptosis. Caspase-3 induces apoptosis by: cleaving ICAD, which then allows CAD to cleave DNA into fragments; cleaves nuclear lamins, which allows the nucleus to condense; and it cleaves PAK-2, which plays a role in membrane blebbing; among other things. DNA fragmentation, nuclear condensation, and membrane blebbing are all hallmarks of apoptosis.
Caspase-3 has been extensively studied with respects to its role in apoptosis. It has been shown that active caspase-3 is expressed in neurons, astrocytes, and oligodendrocytes. It has been demonstrated that active caspase-3 is present in the brain during the early stages of development, but present in only limited amounts in postmitotic neurons in later developmental stages.
Recent studies in the past few years have shown that there are dual roles for some of the caspases. Caspases have been implicated in a number of non-apoptotic roles such as cell survival pathways, cell motility, cell cycle regulation, proliferation, and differentiation. Of the dual roles studied thus far for caspase-3, it appears to be a common phenomenon for caspase-3 to play an essential part of cellular differentiation.
In our lab, we use Cell Signaling Technology's Cleaved Caspase-3 (D175) Antibody to look at the possibility of active caspase-3 serving a non-apoptotic function in the cerebellum, possibly involved in proliferation and differentiation of the EGL cells as well as Bergmann glial cells. We use Cell Signaling Technology's Cleaved Caspase-3 (D175) Antibody because it detects only the large fragment of activated caspase-3 (17-20 kD). It does not recognize endogenous levels of full length caspase-3 or other caspases.
Cell Signaling Technology's Cleaved Caspase-3 (D175) Antibody is a polyclonal antibody that cross reacts with human, rat, and mouse caspase-3. It can be used for western blotting, immunocytochemistry, and immunohistochemistry. We have used this antibody in our lab for all three applications. It is suggested to use this antibody for immunocytochemistry and immunohistochemistry (which are the main two applications used in our lab) at a 1:100 dilution, although after conducting several dilution curves we have found a 1:50 dilution to be optimal for our studies.
Our lab has tried a number of caspase 3 antibodies and we have found Cell Signaling Technology's Cleaved Caspase-3 (D175) Antibody to give the best results. We use our immuno procedures to conduct fluorescent confocal microscopy, and have found Cell Signaling Technology's Cleaved Caspase-3 (D175) Antibody to give an excellent signal in the EGL and the Bergmann glial cells of the cerebellum as well as glial cultures, especially when it is used in conjunction with Molecular Probes' goat anti rabbit Alexa Fluor 488 and 594 secondary antibodies.
We have been using this antibody for over three years now and have been extremely pleased with it. It has given us consistent results that we have been able to replicate time and time again. Of course, at $600.00 for 300l, it can get quite costly very quickly, especially with a 1:50 dilution and using 250l per sample (which our lab did for a long time, until we came across RPI's coverwell incubation chambers). The cost is enough to turn anyone away, but once the results are obtained, it makes it all worthwhile. I would definitely recommend using Cell Signaling Technology's Cleaved Caspase-3 (D175) Antibody to anyone who is doing apoptotic/non-apoptotic studies of caspases.
VelvetLee Finckbone, MS
Dept. of Physiology
Texas Tech University Health Science Center