To validate that the quaternary association of IPO is also a tetrameric form in solution, purified IPO was used in gel filtration experiments. The molecular mass of IPO could be calculated according to the linear regression equation of the standard protein markers purchased from BioRad ( Figure 4C ). In the preliminary study, IPO protein was dissolved in running buffer (27 mM Tris-HCl pH 7.0, 2 M NaCl) without additional carbohydrates. We obtained a retarded result, with corresponding molecular mass 4.0 kDa (Peak 3 in Figure 4A ). Thus, IPO has the binding ability of dextran in the matrix of the Superdex 200 column. To eliminate the binding effect of IPO to dextran, running buffer was prepared with an additional 0.2 M Me-Glc, and a shift of the IPO peak could be observed, with corresponding molecular mass of 53.3 kDa (Peak 2 in Figure 4A ). Consequently, running buffer with an additional 1 M glucose was prepared to totally eliminate the binding effect of IPO. The corresponding molecular mass of IPO in solution was 64.7 kDa (Peak 1 in Figure 4A ). The molecular mass of recombinant IPO with a His tag was 17.3 kDa for a monomer and 69.2 kDa for a tetramer. The results from gel filtration experiments demonstrated that IPO shows a tetrameric association in solution.