As long as your reagents are not specified in more detail, it is difficult to identify the problem. However, according to my experience, any partial denaturation by chaotropic reagents would make it worse. Sometimes the addition of BSA or other proteins might help. If your background is caused by an unwanted specific interaction to your polymer, non-specific blocking will fail in any case. The most successful approach to reducing the background in peroxidase-based assays is to dilute the peroxidase reagent.

Thank you for your response, Michael. To dilute and to wait longer during the substrate reaction may be a good idea. My question is about "naked" PVA with no specific interactions with the conjugate, which, however, shows some binding. BSA typically helps with polystyrene plates to reduce hydrophobic interactions, while here I have a well hydrated polymer.    

We see improvements with BSA in the buffer at different kinds of surfaces. Proteins can make all kinds of interactions, not only hydrophobic ones.

In addition, it is known that PVA and starch (carbohydrate!) can form strong complexes. Perhaps the carbohydrate of the peroxidase or of the antibody may cause the problem here.

Finally, the hydrogel structure itself might be the problem. Reagents entering the gel might need a very long time to exit the gel by diffusion during the washing steps.

Sasha, 

I need experimental details, "the devil in details".

Nicolai,

nice to hear from you ! The devil is also that I can not disclose many details :) It seems like I removed all the activated functions after cross-linking. Thus it is the  hydroxylated matirx that plays role. Imagine you have this kind of binding on Sepharose, what you would do ? Thank you for your attention. 

Sasha,

Is it purified protein, serum, or something else?

Bests

K

Kolya,

this is a purified IgG-peroxidase conjugate that non-specifically adsorbs to the "naked" gel from PBS. I would like to reduce this adsorption. Regards

S