FIGURE 3.

Premixing of TSG-6 and IαI affects protein incorporation. A, co-incubation assay by ellipsometry. TSG-6 and IαI were premixed for 120 min prior to exposure to the HA (837 kDa) film at final concentrations of 0.3 and 1.0 μm, respectively (filled triangles). Alternatively, first IαI and then TSG-6 were added to the HA film (open circles). The two premixing scenarios led to distinctly different binding and unbinding responses. The dashed lines are linear fits to the data at the start of incubation, giving initial binding rates of 1.8 and 1.1 ng/cm²/min, respectively. B, Western blots of the fractions eluted with 2 and 8 m GuHCl and of HA-ase digests. The numbers at the top of the lanes correspond to the premixing time in minutes; assignments of the strongest bands are indicated. Western blots for the control reaction mix (CRM) of TSG-6, HA₁₄, and IαI are also shown. b-HA, biotinylated HA.