FIGURE 2.
Monomeric MD-2 can be isolated in good yield on Sephacryl S200 using insect medium, but not PBS, as eluant. A and B, concentrated (10×) conditioned insect medium was applied to Sephacryl S200 (1.6 ×∼70 cm) equilibrated and eluted in PBS (A) or Express Five™ medium (B). Aliquots (25 μl) of individual fractions (1 ml) were resolved by SDS-PAGE under nonreducing conditions and after transfer to nitrocellulose probed for MD-2-His by reactivity with an anti-(His)4 antibody. C, fractions from Sephacryl S200 eluted in Express Five™ medium (B) corresponding to dimer and monomer forms of MD-2 as determined by the immunoblot were pooled. Pooled fractions of dimer (lanes D; fractions 73–77) and monomer (lanes M; fractions 92–96) and concentrated conditioned medium (lanes CM) were treated with sample buffer ± dithiothreitol, electrophoresed, transferred to nitrocellulose, and then probed for MD-2 using an anti-(His)4 antibody. The blots shown are representative of fractions derived from two different chromatographic separations for each eluant. Note that testing of fractions shown required two separate blots; however, the gels were electrophoresed, transferred, and probed with anti-(His)4 antibody simultaneously.