Positive dot blot, negative WB

[durante]

Hi Guys!

It is an honour to be a member of this forum. I hope to share my (little) scientific experience with all of you.

Well, I am frustrate by my WB since I am not getting any results. I am searching for an aromatasis enzyme (p450C17) on brain homogenates. The protocol I use it's validate since I worked with many other protein without any problems and I can exclude major technical problems at least until the incubation in the primary antibody (Ponceau staining is positive and the run is apparently good). But at the end of all process my membranes are completely white (the positive control is negative as well!)
The strange thing is that when I performed Dot-blots using the same samples, same positive controls, same antobodies at the same concentrations I got positive results. So, at this time I have no solution. In the meanwhile I am performing IF on slices of the same brain regions and I'll see. I am planning to perform an immunoiprecipitation before WB or change something in WB protocol but I am not sure that it could work.

I appreciate any suggestions/explanation from all of you.

Thank you!

paolo