Recombinant ZC3H11 Protein Purification

Fragments of the ZC3H11 open reading frame (first 104a.a., 119a.a., 136a.a.) were cloned into pQEA38vector after His₁₀-tag, between KpnI and HindIII sites. Bacteria (E.coli strain Rosetta pLysS, Novagen) were grown at room temperature to an OD600 of 0.6, induced with 0.25mM isopropyl β-D-1-thiogalactopyranoside and incubated at the same temperature for five hours before harvesting. Recombinant proteins were purified with Ni-NTA Agarose (QIAGEN) following the manufacturers’ instructions [20 (link)]. Buffer in protein samples was exchanged to PBS. Rabbits were immunized with His₁₀-ZC3H11 (119a.a.) according to standard procedures (Charles River Laboratories, Kisslegg, Germany). Polyclonal antibodies were affinity-purified from crude anti-serum using His₁₀-ZC3H11 (119a.a.) fragment coupled to CNBR-activated Sepharose (GE Healthcare).