One of my colleague is doing western blotting, and developed it with ECL on X-Ray film, but getting heavy background along with bands even after increasing washing with TBST and concentration of secondary antibody.
What else do you have in your TBST as block? We usually include either Blotto (milk powder) although this cannot be used with anti-phosphate antibodies or you can add 2% BSA to your TBST which can also be used with anti-phosphate antibodies.
Modify the blocking solution. Your antibodies are binding all over the blot and giving you a background. Try casien or BSA instead of milk.
I agree with those who suggested to modify the conditions for blocking.
Beside all been said earlier, check if your target protein has phosphrylated sites or is subject to other post translational modifications such as trnasmemebrane demains ormembrane insertion leading Is the antibody being used home brew or commercially available? If you
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