Figure 2.

IgG-binding activity of r0375. (A) SDS-PAGE identifies the IgG-binding activity of r0375. M, Molecular mass marker. Purified r0375 is conjugated to the nickel agarose via its histidine tag, and incubated with non-specific IgG from cattle, swine, horse, goat, mouse, rabbit, chicken, human. After washing, IgG bound to the agarose is detected by SDS-PAGE. The control group includes the nickel agarose conjugated r0375 and the nickel agarose incubated with IgG. (B) Western blotting identifies the IgG-binding activity of r0375. r0375 and rP48 were separated by SDS-PAGE and transferred onto PVDF membrane, then HRP-conjugated IgG of goat, or mouse was incubated, respectively. IgG bound to the membrane is detected. (C) ELISA identifies the IgG-binding activity of r0375. Bovine or goat IgG (5 μg per well) were coated on the ELISA plates, PBS or BSA (5 μg per well) were coated as controls. The plates were incubated with r0375 at different concentrations. Using mouse anti-His tag IgG as the primary antibody and HRP-conjugated goat anti-mouse IgG as the second antibody. The value of optical density was measured with TMB as substrate. (D) Rabbit IgG (5 μg per well) was coated on ELISA plates and other procedures are the same as described in (C). Error bars correspond to the SD of three technical replicates.