overall:I ran a western on eluate from an IP. (3 gels). I ran eluates on Bio Rad TGX gel in 1X Tris glycine running buffer. Transfered in 1x Tris glycine Transfer buffer with 15% methanol, (using dH2O), using the wet transfer method. Post transfer, I washed 15 minutes in TBST, blocked 1 hour in 2% milk in TBST, and probed overnight with monoclonal AB of interest. Washed and used goat anti-mouse-HRP. I detected using regular ECL.

Results: (for the sake of simplicity I have included a representative example of four of the bands from 1 blot. What happened with these for lanes was true for the most part with the all of the bands from 3 blots).

First time,---all four bands were about same intensity. (see attached figures). This was not what I expected as two lanes were positive controls that had worked in the past. I stripped the blot for an hour with mild stripping buffer and reprobed with the [AB in milk] that I had saved from the previous probe. In this second probe---the two positive control bands were darker than the untreated, as expected.

So it seems that something was interferring with AB from fully interacting with some of the protein that there on the blot-, as if there was a covering or interferring agent of some type that was removed with the stripping. Has anyone seen this before: and/or can offer possible explanations? I use regular methanol, . Perhaps I need more pure methanol. I use dH2O.