Generation and characterization of a polyclonal antibody against human high mobility group box 4.

A human high mobility group box 4 (hHMGB4) expression construct (pET‑28a/hHMGB4) was generated by cloning the hHMGB4 full‑length cDNA in the expression vector pET‑28a(+). The hHMGB4 fusion protein with His6‑Tag was prepared using E.coli BL21 (DE3) transformed with pET‑28a/hHMGB4 and purified via preparative SDS‑PAGE plus electroelution. Immunization of rabbits with the purified hHMGB4 generated polyclonal antibodies. The titer of the antiserum was determined to be 1:102,400 by ELISA analysis. Western blotting analysis showed that the antibody specifically recognized the recombinant hHMGB4 protein and also the endogenous hHMGB4 protein in prostate cancer cells. In addition, immunohistochemical staining analysis using the prepared antibody revealed marked hHMGB4 staining in the nuclei of the human prostate tissue. These data demonstrate that the anti‑hHMGB4 polyclonal antibody may be a useful reagent for the functional study of hHMGB4.