Hi everyone,
I'm not able to detect cleaved caspase-3 by western blotting using the ab13847 antibody. My wb input are lysed primary cortical neurons treated with staurosporine 1 uM for 4 and 6 h. I've tried to load 30, 40 and 50 ug of total cell lysate in 5-12% and 5-15% (stacking-separating) gels. I'm blocking the membrane using TBST-5% skimmed milk and the primary dilutions is the one recommended by the manufacturer (1/500). I've also tried different wet transfer (Tris-Glycine-Methanol) conditions: 100V 1 h and 100V 30'. The antibody only detects the 32 kD band corresponding to inactive caspase-3, even after 30' of exposition.
Can anybody give me some advice in order to detect the cleaved-caspase 3? Thanks in advance for the attention.
Hi!
Your antibody can detect p73 not cleaved caspase 3. You can try this one : https://www.cellsignal.com/products/primary-antibodies/cleaved-caspase-3-asp175-antibody/9661
Have a good day
You are probably talking about ab13847, not ab31847 (which is used for p72). In my apoptosis studies, I used an antibody from Cell Signaling (#14220), which always worked fine (1:1.000 in TBST + 5 % BSA).
https://www.cellsignal.com/products/primary-antibodies/caspase-3-d3r6y-rabbit-mab/14220
But you should also consider to increase the staurosporine concentration or to switch to another positive control, e.g. etoposide
Good luck, CL
Hi,
I would recommend the cleaved caspase 3 antibody from cell signalling, it worked well for me on gradient gels
https://www.cellsignal.com/products/primary-antibodies/cleaved-caspase-3-asp175-antibody/9661?site-search-type=Products
On the other note, I am not sure how long are you running your gel? I think 1h transfer is too long for cleaved caspase bands as they range from 12-20kDa and are small. Make your your dye front of the gel is not running out of the tank. Try with stopping it say 1 cm before it runs out of the gel.
Good luck
Hi everyone!! I've tried the cell signaling antibody (#9661) and it is amazing, it worked well at the first try.
- Badges
- Science method