Hi everyone!
I did immunostaining of sham and stroke tissue with Total caspase 3 ( Caspase-3 Antibody #9662), and I could able to see staining in sham animal tissue too. So, is it normal to see total caspase in without disease group?, if yes then what is that significance of doing that instead of cleaved caspase 3? Or it is just for the comparison purpose between total and cleaved caspase 3?
Thanks,
Dear Amisha,
in my opinion it is quite normal, that you see Casp3 in both samples. Your antibody stains pro-caspase3 and the cleaved form.
You will finde pro-caspase3 in all cells for all time, because the cell wants to be prepared for apoptosis if necessary.
Best regards
Franz
Dear Amisha,
yes, it is for the comparison purpose - to compare between total and cleaved caspase 3. The total caspase-3 ( or pro-caspase form) is used as loading control in western blots. It SHOULD be present in the tissue/cells before treatment (unless caspase in down-regulated in cancer cells like MCF-7).
The specific treatment might induce apoptosis and cleavage of the pro-caspase-3. than you need to estimate how much caspase (using image J or other software) you had in cell before treatment and how much cleaved caspase you had after treatment, and find a ratio between two. see below an example - figure 8 in the attached paper - represents a proper experiment.
To figure out whether caspase 3 pathway was activated you have to check cleaved form of caspase 3. Total caspase 3 will say nothing about activation of apoptosis pathway.
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