Hi all,
I have treated the drug that induces DSB to the cells (HEK293T). After 24 h of incubation, I harvested and did Western blot for gH2AX but I did not see any bands. I made 12% gel and loaded 30 ug protein. Do I need to change my routine WB protocol?
Also, I would like to keep a positive control Is that fine if I keep the cells under UV of the clean bench for some time and followed by WB? Because I don't have the irradiation equipment.
P.S. I am using Millipore gH2AX (05-636)
Please shed some light.
TIA.
Arun.
There could be few reasons of that. Firstly - maybe your drug in used concentration do not cause DNA damage at all. You must use positive control to see whether this is the case. Secondly - maybe the blocking procedure is too strong. Sometimes blocking in 5% non fatty milk enables detection of weak bands. Try in 5% BSA instead. Thirdly - maybe it is the wrong antibody. To check that you also need the positive control. If positive control do not have the band, then it is bad antibody. You can also stain treated cells with gH2AX to check whether or not there are foci of damaged DNA within nucleus. As a positive control you can use cells treated with drug that is proven to cause DNA damage, for example Doxorubicin. Using UV light can be tricky, because same cells are more resistant that others. I hope that will help :)
Agree with agnieszka, i use BSA 5% 1H blocking condition.
you have to validate the antibody to move forward.
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