Hello dear researchers,
I would like to know whether there is someone who has been successful in detecting active-caspase3 and 9 and cleaved PARP using western blot.
I would be really grateful for those who will share their experience(transfert time,transfert buffer composition, loading protein amount, cells harvest time etc).
Thanks in advance.
Hamadi
I have detected PARP (full and cleaved forms) successfully.
The conditions that I used:
- 100 volts for 1 hour using Tris-glycine buffer with 20% methanol
- nitrocellulose membrane 0.45 um
- anti-PARP Ab (clone C2-10) 1:1000 dilution
I've detected full length and cleaved caspase 3. The conditions were:
- 40 ug protein, 12% SDS-polyacrylamide gel
- Transfer buffer Tris-glycine + 20% methanol
- 90 min, 250 mA
- 0.2 um-pore nitrocellulose membrane
- Caspase 3 antibody (#9662 Cell Signaling) dil 1:1000
- Secondary antibody: HRP-conjugated polyclonal goat anti-rabbit (P0448 Dako) dil 1:2000
- Positive control: Staurosporine treated cells
Hope this helps!
Hi Hamadi, what type of cells are you using (mouse, rat, human?). If it is mouse, all the information is a paper from my group http://www.ncbi.nlm.nih.gov/pubmed/25470234 .
A lot of these antibodies will work of different species, just check the datasheet.
Hi, I am looking for caspase 3 cleavage in leukemia cell line which are floating cells, so what will be the condition for western blot.
Thank You
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