Donkey anti rabbit igg secondary antibodies conjugated to horseradish peroxidase

Sarkosyl-insoluble material was purified from 150mg of AD postmortem brain tissue and resuspended in 250uL TBS. Recombinant AD tau core (polymerized or monomeric) was diluted to 0.125ug/uL. 40uL of each sample was incubated in the presence or absence of 0.4mg/mL pronase (Millipore) at 37°C. Protease inhibitor cocktail was then added to stop the reaction at either 1, 2 or 5 minutes as indicated. pronase-treated and untreated samples were then spotted onto a nitrocellulose membrane to evaluate immunoreactivity for ADC1 and E1 tau antibodies. Briefly, dot blots were washed in TBS plus 0.1% Triton-X (TBS-T), and incubated overnight at 4C with the indicated primary antibodies. The following day, blots were washed in TBS-T, incubated with donkey anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase (1:5000; Jackson ImmunoResearch) for 1 hour, and again washed with TBS-T. Protein expression was then visualized by enhanced chemiluminescence treatment and exposure to film.