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
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 Possibly the antibodies are out of date?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?