Ghost and inverse bands and high background. Help! - (Oct/11/2012 )

I'm almost at my wits end with trying to get my western blots to work...

I keep getting ghost bands (inverse/white bands) where my protein should be. Also a very high background.

<*>My proteins are at 25ng
<*>After running the gel I use Thermo scientific Superblock (T20 TBS) blocking buffer for 1- 1.5 hours, 12mls
<*>Add 1:10,000 primary antibody. (I've used c-Jun, Galectin-3, Akt, JNK all from abcam)
<*>4degC on a rocker overnight
<*>Wash x2 in TBST, then rinse in TBST 3x 5mins (sometimes maybe longer i.e total 30mins)
<*>Add secondary antibody 1:10000 to 12mls TBST, 1 hour
<*>Rinse and wash blot 3x 5mins
<*>I use Supersignal west pico ECL. I blot the blot, then add 3mls on blot for 5 mins
<*>Blot off the ECL, and image 1-40secs

I even stripped one blot yesterday that had ghost bands (cJun), and then added B actin antibody. Nothing came up on the image today

Any ideas at all I'd be grateful as we're a bit stumped! Possibly the antibodies are out of date? (2008). Although the B actin antibody should work fine.
Am I washing too much? Too much protein? Too much antibody?
Thanks!

Ghost bands often appear when you have way too much antibody, as the HRP rapidly depletes the ECL reagent in the region of your bands. Also adds to the high background. I would try even less antibody and you could also try using a less sensitive ECL reagent.

And you're definitely not washing the blots too much. 3x washes is the absolute minimum number of washes I would do to get a nice blot with low background, so I would add a few more washes as well.

load more and get fresh antibody unless those were aliquoted and frozen

Hi thanks for the replies. I actually found that my TBST stock was at the wrong pH - was at pH4! So I'm made it fresh. I repeated the B actin and it worked perfectly, but I also repeated Galectin-3 and it didn't - still got ghost bands. I will try much less antibody today - maybe 0.8ul in 12mls.

We were worried that maybe the antibodies just don't work anymore. I aliquoted and froze the Galectin-3 antibody, but it was aliquoted at 4ul - so each one has been taken out 3-4 times. All the other antibodies I've used are kept in the fridge and not aliquoted...