Anti mouse igg bp hrp

Human anti-PrP antibody D13 (1:400), which recognizes mouse PrP, was dissolved in CB and used to coat the wells of a 96-well microplate at 4 °C overnight. Between all steps, the plate was washed with PBST. Wells were blocked with BSA-PBST at room temperature for 1 h. Serial dilutions of recombinant PrP (Invitrogen) in BSA-PBST were added to the wells to generate a standard curve. The 10% brain homogenates from WT (FVB), TgPrP^−/−, TgGSS and TgHuPrP mice were serially diluted in BSA-PBST and added to wells of the plate and incubated overnight at 4 °C. Mouse anti-PrP mAb SAF-32 (SPI bio) (1:400) was then added, followed by anti-mouse IgG BP-HRP (Santa Cruz) (1:2,500) at room temperature for 1 h. After incubation with TMB at 37 °C for 5–20 min, 2N HCl was added to stop the reaction and read on a plate reader at O.D. 450 nm to calculate PrP concentrations.

The synthesized human Aβ42 peptide (GenScript, NJ) (1,400 nM) was dissolved in CB (50 µl) and added to wells of a 96-well microplate and incubated at 4 °C overnight. The plate was washed with PBST between each step. The wells were blocked with BSA-PBST at room temperature for 1 h. Serial dilutions of recombinant human PrP (Invitrogen) dissolved in BSA-PBST were added to the wells to generate a standard curve. The 10% brain homogenates from WT FVB, TgPrP^−/−, TgGSS, and TgHuPrP mice were serially diluted in BSA-PBST, added to wells and incubated at 4 °C overnight. Wells were then incubated with mouse anti-PrP mAb SAF-32 (SPI bio) (1:400) followed by the anti-mouse IgG BP-HRP (Santa Cruz) (1:2,500) at room temperature for 1 h. After incubation with TMB at 37 °C for 5–20 min, 2N HCl was added to stop the reaction. O.D. at 450 nm was measured to calculate the binding.