Figure 6
From: Recombinant Human Prion Protein Inhibits Prion Propagation in vitro
Binding of recombinant human PrP23-231 to human PrPSc.
(A) Capture of PrPSc or PrPC by rHuPrP23-231 was performed by incubation of rHuPrP23-231-conjugated magnetic beads with uninfected (CTL) and iCJD human brain homogenates, respectively. Magnetic beads without conjugated proteins were used as negative control (Empty). The g5p-conjugated beads were used as a positive control (g5p). To determine the specificity of the binding, we also examined the beads conjugated with recombinant PDI. The PK-resistant PrPSc was only detected in the preparations captured by rHuPrP23-231 and g5p beads from CJD brain homogenates. In contrast, no PK-resistant PrP was detected in CJD samples captured by empty beads and PDI beads. No PrP signal was detected in the uninfected samples captured by all the beads except the rHuPrP beads. The bands detected in the preparation captured by rHuPrP beads are expected to be the recombinant PrP itself. (B) To determine whether PrP detected in the preparation captured by rHuPrP from the uninfected brain homogenate shown in panel (A) was brain PrPC or rHuPrP itself, the capture experiment was also performed in the binding buffer alone without uninfected brain homogenate. The virtual same PrP bands were detected in both capture experiments in the absence and presence of uninfected human brain homogenates, suggesting that the detected PrP bands were from rHuPrP itself and no brain PrPC was captured. The blots were probed with 3F4. The results shown in (A) and (B) are a representative of two experiments.
