Dot blot: dots on film bright white/clear not black?

[cardiffpHD]

I am trying to dot blot for an antigen in cell lysate that has been fractionated (using optiprep). I used the abcam protocol that I have used on cell lysate before. My antigen control works fine (black dots, good concentration range), but some of the fractions appear clear on the film (against a greyish membrane background). Why is this? It is the same fractions each time, but not all the fractions - I'm not sure whether the concentration is too high or there is something in the lysate that could inhibit the antibody binding?

Can anybody shed some light? Thanks

[cintiadp]

Probably your secondary is too high or the concentration is too high that is consuming all your enzymatic reaction. Try diluting out your fraction or the secondary antibody. I hope that works.
Cintia