Performed multiple western blots on two different cell lines with several primary antibodies used. Loading control has been successful for all blots. No bands for any lanes.
If I understood correctly, when you load pure (commercial?) protein and transfer, you observe band, but on the same gel/blot you see no signal with your extract?
Can you check expression of your genes by qPCR? Just to make sure.
Maybe try to decrease amount of control as much as possible to enable you for longer exposition, so that you can detect even minute amounts of your POI in your extracts.
Your protein may not be abundant enough to detect on western blot. What type of secondary antibody are you using? If HRP conjugated you can try a higher sensitivity substrate, or you may want to try immunoprecipitating the protein before western blot.
In my mind, you may add some positive samples to evaluate the effectiveness of the western blot. You can optimize detection conditions, such as sample volume， antibody concentration, and incubation time.
Good luck
Guosong
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