Western blots - positive control - (Apr/24/2007 )

I am carring out western blots. all bands was NOT detected included positive control.
So I confirmed several things.

( positive control : purified protein)

1. purified protein : measure its quantity and its activity

2. dot blot : No problem - 1st Ab.

If you have know-how, help me ~~^^ please~~^^*

-Have you loaded enough total protein?
-Do you know if 2nd Ab is working? (are you using it in others Westerns?) or if its dilution is appropiate (may it be too diluted?)?
-Are you using ECL?: is it fresh? Did you try several exposure times?