Western didnot work should I strip blot if i want to detect same protein-Notch I - (Apr/06/2011 )
Hi, Am a novice at westerns and would appreciate any help I can get. I am trying to detect the Notch1 Intracellular domain from mouse embryo tail buds (10.5 days postconception). I use a popular Cleaved Notch antibody from Cell Signaling. I ran multiple westerns unsuccessfully (everytime it was a different issue- either I used too much tween or the secondary was too high- I had high background- black blots or no signal at all) and now am all out of samples. I have my blots both PVDF and nitrocellulose dried as well as some stored in TBST. I wanted to try reprobing some of them for the same protein (before accepting defeat and turning the whole thing over to my superior colleague). My question is should I just wet the membranes (in case of dried ones) and start from secondary antibody incubation step or should I strip the blot and reprobe? I am sure I am loading enough protein - when I get black blots, I see white bands and I was told thats due to protein overloading.
Reprobing rarely works like it is supposed to, so I'd steer clear of that. Otherwise it depends on what exactly went wrong the first time. The antibodies more or less are still there on your membrane, so odds are you will get the same thing again.