Did you immunize against denatured protein (e.g., a band cut out from a SDS-PAGE gel)? In that case, your result would be a common one.

- If you want antibodies against native proteins (e.g., for immunoprecipitation) inject the native protein.

-If you want antibodies against denatured protein (e.g., for western blot), inject the denatured protein.

Sometimes you get lucky and a serum works for both, but there is no guarantee.

When you say your antibody did not detect native proteins, how did you test it? Did you run a native PAGE and native western blot or do you mean by that just a whole cell extract on SDS PAGE?

Do you know whether your protein is expressed in sufficient amounts? You could test by qPCR for relative expression on mRNA level (to have a rough estimate whether it's high or low expressed). Also, do you know of any conditions in which your protein might be upregulated, so you could test those as well for western blotting.

Is the protein posttranslationally modified? In the worst case in your cell extracts the PTMs mask the epitopes for the antibody (although I think it's unlikely that for a polyclonal antibody all epitopes are masked).

You could also try to either purify or enrich the antibody.

A final suggestion would be to couple the antibody to protein A or protein G beads and do an IP against recombinant protein and whole cell extract (native and denatured) to see whether the antibody can generally bind the protein / whether you can enrich it.

Thank you for your recommends.

@ Engelbert Buxbaum I didn't immunize against denatured protein, but I will try as your suggestions.

@ Sascha Röth I collected the antibody from mice (mice was injected by my recombinant protein), then I run western blot with recombinant protein and whole cell extract, using antibody I made as first antibody. But after developing, antibody only detected recombinant protein.

I also checked the expression of my protein by RT-PCR and they expressed well. and you mean my cell extracts the protein posttranslational modifies masks the epitopes fro the antibody. How can I know whether my protein was modified or not?

Try immunoprecipitating the target protein from a larger amount of amoeba lysate and then detecting it by Western blot.