Figure S4.

Characterization of GP-ectoΔmucin and GPcl Proteins and Binding Affinities of 2G4 Bound to GP and GPcl, Related to Figure 2

(A) The GPectoΔmucin protein was eluted as a single peak (the elution volume represents a molecular weight of ∼180 kDa, homotrimer) in the gel filtration of Superdex^® 200 column (GE Healthcare). The primed GP protein (GPcl) was also eluted as a single peak (the elution volume represents a molecular weight of ∼120 kDa). In the SDS-PAGE, after thermolycin digestion, the GP1 subunit was cleaved into a 19 kDa band, the molecular weight of which is similar to the GP2 subunit.

(B) The GP-ectoΔmucin and GPcl proteins were immobilized on the CM5 chip with about 2000 response units. Then serial concentrations of 2G4 Fab were flow through the chip. The BIAcore^® diagrams reveal that 2G4 Fab binds to GP or GPcl with slow on and off rates. Moreover, 2G4 Fab binds to GP or GPcl with similar binding affinities (8.2 nM versus 3.8 nM). The K_D values were calculated by the BIAcore^® 3000 analysis software (BIAevaluation Version 4.1).