I need suggestions, as my blots are too dark(high background) I can see my bands are weak hidden under dark background. MY blotting condition are
Transfer condition- 80 volts-2hrs (tris buffer 25mM, 192mM glycine 20% methanol)
Blocking buffer- TBST (0.1% tween20) with 5%milk powder- 2hrs
Primary antibody- 1:200 dilution in blocking buffer- overnight
Wash 3times 10min each in TBST
Secondary antibody- 1:3000 dilution in blocking buffer- 1hrs
wash 5 times in TBST and 1 time in TBS 10min each.
Exposure-ECL reagent (Luminol)..
The background is too dark..please suggest your comments.