I have some problem with reprobing my membrane after stripping.
After ECL detection, I directly put my nitrocellulose membrane in PBS. After rincing the ECL, The membrane are incubated with stripping buffer (TRIS 62,5 mM-SDS 2% - Mercaptoethanol 100 mM - pH 6,7) 30 min at 50°C under gentle agitation. After, membrane are washed several times with PBS until no odour of mercaptoethanol can be smelt. Then, membrane are reblocked and reprobed with primary antibody overnight at 4°C.
The first probing works very well (detection of the phosphorylated protein). It is when I want to detect the non-phosphorylated protein on the same blot (after stripping) that I get no signal. There is no problem with the antibody because when I first try to detect the non-phosphorylated protein, it works.
If somebody could help me, it would be grateful.
Thanks in advance