Hi all,
I am currently studying activation of PARP after inducing DNA damage. The method I follow is:
1) exposing tumor cells (HCT116, HeLa cells) and MEFs (WT and S12) to DNA damaging agent Temozolomide for 1 hour and then the removal of the media containing Temozolomide followed by treatment with a PARP inhibitor like Olaparib
2)protein conc. estimation by BCA assay,
3)separation by SDS-PAGE,
4)overnight wet transfer and
5)immunoblotting with PARP 10H mouse monoclonal antibody from Genetex (1:250 dilution) 24-48 hours 4 degrees C and Rabbit anti-mouse IgG3 HRP (1:250 dilution) followed by chemiluminiscent detection by Supersignal substrate
The principle of the whole method is that PARP production is higher upon treatment with a DNA damaging agent like Temozolomide and minimized upon treatment with a PARP inhibitor like Olaparib.
The issue I am currently facing is: the PARP production is less even with DNA damage by TMZ as opposed to a control without any TMZ/Olaparib. A higher concentration of a test PARPi is sometimes exhibiting less PARP inhibition as opposed to a 10-fold lower concentration of the same. Attached is an image depicting the same. Kindly provide any suggestions to rectify the issue.
PS: This doesn't happen all the time. In some western blots, the results conform the hypothesis perfectly.
Hi
Have you done any annexin v staining to see if temozolomide causes apoptosis?
Temozolomide mainly causes cell cycle arrest rather than apoptosis so it is not unusual to see no PARP cleavage with temozolomide.
Maybe you can try another DNA damaging agent such as doxorobucin that causes apoptosis and PARP cleavage as a positive control.
Regarding the blot attached, is this total PARP shown or cleaved PARP? Could be that total PARP is cleaved but antibody is not sensitive enough to detect the cleaved fragments.
It seems that the antibody you are using detects PADPR. PADPR formation is catalysed by PARP. So maybe that's why you see a decrease in band intensity with PARP inhibitor. A higher conc of PARPi will cause less PADPR formation and weaker band on your blot
I agree with Fadi Gurgis, you need to know if your antibody is detecting the PARP protein (cleaved or uncleaved), or the poly ADP-ribose chains (PADPR) ribosylated by the PARP enzyme and hence, an indicator of PARP activity. PARP inhibitors like olaparib prevent PARP activity i.e. ADP-ribosylation, which is why you see a decrease in your band intensity with increasing PARPi concentration.
Alternatively, you could use hydrogen peroxide (20mM, 15min at RT in the dark) to stimulate PARP activity, and immunofluorescent staining and microscopy to visualise/quantify.
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