I tried to detect ATM protein by western blot without any success.

Have anyone experience with it?

I tell you my protocol:

Cells with which I work are MRC-5. I use a cell total lysis buffer.

I perform electrophoresis with handmade polyacrilamide gels, 3% stacking and 7% separating gel. The running buffer used is Tris-Glycine.

I have tried different protocols for transfer: with and without methanol, with and without 0.1% SDS. I always do a wet transfer in the Mini Trans-Blot cell tank (Bio-Rad). I test 70V 3h, 100V 1h and O/N 15-20V for about 16 hours. However, when I stained the PVDF membrane with Ponceau , I do not see any signal above 250 kDa.

Any suggestions?

Thank you in advance