Cannot figure this dot blot thing out - (Apr/05/2016 )

I'm trying to dot blot with PVDF.  I prewetted the membrane with methanol for 30 secs then equilibrated in NuPage transfer buffer for ~5 min (also contains 10% methanol).  I then poured off transfer buffer, added 2uL protein sample, and let the membrane dry.  According to all protocols I've read, this is correct.  I then went on to block and stain with antibody (TBST in 3% BSA per the antibody manufacturer's instructions).  The problem is the blot looked dry throughout all these steps and never rehydrated (stayed white entire time).  To detect, I added ECL (1:1 Pierce femto staining kit), let sit on membrane for ~3 mins, wicked off excess and imaged.  These resulted in extremely high background across the entire blot.  So I rinsed it under water once and reimaged.  I was able to detect a vague dot in my positive control very briefly (enough to capture a 'good enough' image) but the chemiluminesce quickly went away (within ~1 min time).  What could I possibly be doing wrong?  Should I have rehydrated the blot somewhere along the way?  Is this easier with nitrocellulose?

PVDF has to be re-wetted the same way everytime after it dries out , ie, methanol, water, buffer. If you did not start the re-wetting process with methanol, than it would not rewet with any subsequent aqueous buffers.

Cheers

however, if you don't re-wet then your antibodies will only be able to react with the spot and not non-specifically with the membrane.