To raise antibodies against ZHP-4 and avoid cross-reactivity with other RING domain containing proteins, a fragment of 372 base pairs corresponding to the C-terminus of ZHP-4 was cloned into two bacterial expression vectors: pGEX-6p-2, containing the GST tag at the N-ter- minus (GE Healthcare) and pET28a (Qiagen), to generate an N-terminal 6xHis-fusion protein. Recombinant proteins were purified under native conditions using anti-GST beads (GE Healthcare) and Ni-NTA matrix (Qiagen) respectively following the manufacturer's instructions. GST::ZHP-4 was used for antibody production in rat and 6xHis::ZHP-4 was used for sera purification (Medimab). ZHP-4 antibody was purified using activated supports according to the manufacturers' protocols (Affi-Gel 10, BioRad).
zhp-4