Relating to my recent question about high background, I ran a new test. I took un-probed pvdf (both 0.2 um and 0.45 um) and activated them in methanol, put them in water for a few minutes, blocked in 10% NFDM for 1 hour, then probed with various concentrations of my secondary antibody, as well as a primary antibody. The concentrations were 1:2000, 1:5000, and 1:7000. Two of them were made in 5% NFDM (I switched over my blocking method to try to decrease the background but had just made new antibody in the 5%) and one was remade in 10% to see if this was the issue. My 0.2 um membranes showed a darker background when treated with ECL than the 0.45 um membrane, even though I did everything the same. I'm confused as to how these membranes can act differently when the only true difference should be pore size? Is there a chance that the changes in NFDM % caused this change? Is it possible for PVDF to react with HRP or something of that nature? 

I attached images of the 0.45 um and 0.20 um for reference.