Conditions: I performed western at various protein concentrations (50ug, 100ug, 150ug and 200ug). I was able to see ATM band (350 KDa) once, in 150ug & 200ug of protein. But my concern is, a lot of protein will be used in a single experiment.

Details of the experiment:

1. I'm using 6-7 % gel for SDS-PAGE.

2. 150-200ug of protein.

3. Resolving at 95 V for more than 3 hours.

4. Overnight transfer on PVDF membrane (methanol activated) at 30 V with normal 1X transfer buffer.

5. Blocking with 5% Skimmed milk (dissolved in 1X TBST), later incubated 14 hours with ATM Primary antibody (SC-377293) and p-ATM Primary antibody (SC-47739) at 4 degrees.

7. Incubation with secondary mouse antibody for 1.5 hours at RT.

Please help me with this one.