Western blot not showing protein - (Oct/13/2011 )

Hi all,

I have doing western blots for a while and am perplexed by some recent results. I quantified all my protein samples by Bradford and loaded them on a gel. When I blotted with tubulin (as loading control) I only saw a bands in 2 of my 5 samples. The others had no bands. I then decided to Ponceau stain my membrane and it showed equivalent loading of proteins in all 5 lanes! I then tried blotting with a second loading control (actin) that other members of my lab use and had the same result...only 2 samples show protein.

What do you think I might be doing wrong? The ponceau clearly shows there is protein in all lanes and equivalent amounts yet when I tried actin and tubulin I see no bands in 3 of my lanes. ps: this is not the first time this has happened!!!

Do the 3 lanes have anything in common? Different treatment, salts, detergents, cell types, etc? If this has happened more than once, is it the same lanes/samples each time?

All the lanes are fairly similar samples - I isolated protein and quantified all 5 at the same time.