2.4 Solubility of FPOX inclusion bodies
Two different methods including traditional urea-denatured method and freeze-thawing method were used to solubilize inclusion bodies of FPOX in urea. In the urea-denatured method, purified inclusion body was suspended in buffer (NaH2PO4.2H2O/Na2HPO4100 mM, tris-HCl 100 mM, DTT 5 mM) with different concentrations of urea (1, 2, 4, 8 M) and stirred for 30 min at room temperature followed by a centrifugation at 12000g for 20 min, and supernatant was analyzed by SDS-PAGE. The solubility of purified inclusion body also studied in different concentrations of DMSO (5, 10, and 20 %). In the second method, the purified inclusion body of FPOX was suspended in PBS buffer at different concentrations of urea (0.5, 1, 2, 8 M) and the suspension was frozen at -20°C and thawed at room temperature, finally, the supernatant was obtained by a centrifugation at 12000 g for 20 min and analyzed by SDS–PAGE[19]. At the end of this experiments the supernatant was dialysis against refolding buffer (20 mMTris, 100 mM NaCl, 10 mM CaCl2, 0.2mM ZnCl2,pH 7.5) at 4°C for 3 hours.