I'm having some troubles with my Western blotting. I'm using nitrocellulose membranes and 40ug of protein per lane. When staining the filter with Ponceau S, I can detect strong protein bands, but after the antibody incubations I can not detect anything on the film - not even with 1:1000 diluted Rabbit anti B-tubulin (1h at room temperature) and HRP Goat anti Rabbit (1h at room temperature) antibodies. I use Nacho's blocking solution (1h at room temperature) and Thermo Scientific West Femto chemiluminescent substrate solution.
What could be the problem?
With PVDF membranes I had a strong signal with the same protein lysates, so there must be something wrong with the blotting or blocking the membrane or with the antibodies.