Hi there,

Is it direct ELISA? If so are you sure you POI is properly adsorbed onto the plastic? Do you use the same antibody with the same detection as for WB?

Hi Dominique Liger , I used an ELISA kit from STEMCELL technologies. It is based on sandwich ELISA.

No, I did not use same antibodies for WB and ELISA. Since my protein is tagged with a HIS tag, I used an anti-HIS tag antibody for WB, while I used different antibodies for ELISA since its a commercial kit.

Hi,

yes, it is possible to detect a protein with Western Blot and nothing in an ELISA (or vice versa), because the antibody (is it the same antibody?) or two antibodies in case of a sandwich ELISA could only recognize an epitope that is not accessible in the native buffer (e.g. because of oligomerization of folded protein, soluble aggregation of folded or or unfolded protein or the protein or epitope is simply inside the folded protein and not accessible on the surface). If the ELISA usually works with the native folded protein, then your preparation might not be folded correctly. Sensitivity of the antibody in Western Blot and ELISA could also be an issue, so you might be measuring in the wrong concentration range (both of target protein and of the antibody).

Not every antibody is suitable for different applications, therefore you should check the data sheet if it is validated for use in Western Blot and ELISA.

Western Blot is more sensitive than ELISA. You will have more specified results with reduced false positivity in WB results