The receptor binding domain of the coronavirus spike protein has been expressed in Escherichia coli. Antibodies were obtained to the purified protein. These antibodies do not recognize the spike protein in the Western blot. A purified coronavirus was used for Western blotting. What is the reason?
Hi Olga! The western blot was carried out in denatured conditions? In case you did a SDS-Page before the Western Blot only lineal epitopes would be present and the protein recognition will depend on the presence of antibodies that recognise these kind of epitopes, losing the recognition of conformational ones. In that case, you can try with a native page or try a dot-blot.
Ivana Reidel
Yes, the proteins were separated under denaturing conditions. Will try to separate proteins under native conditions. Thanks.
hi
if purified Mabs ok in Elisa test ( against recombinant & wild type virus )
check your Mab in 3 form ( mouse ascites , hybridoma culture supernatant and purified antibody 10 ug/ml )by recombinat protein 100ng & wild virus lystae 20 ug in WB .
good luck
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