Abeta (1-11) | Amyloid-Beta peptide 1-11 (high affinity to fibrils)
AS19 4310 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Human
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All samples was prepared and handled on ice. Monomers of Amyloid-Beta 1-40 and 1-42, recombinant and synthetic peptides respectively (Alexo-Tech AB) were prepared by solubilize 1mg peptide in 50µl 20 mM NaOH followed by dilution with 1x PBS. Finally the peptides were centrifuged to discard any aggregated peptide. For oligomers, the dissolution described above was performed followed by incubation at 25°C for 2 hours with gentle agitation. The oligomer fraction was separated an collected by gel filtration. For fibrils, the dissolution described above was preformed followed by incubation in 37°C for 48h. Before use, the fibrils was sonicated for 2x 1 seconds at 21% amplitude. For all samples, 2µl was dotted on nitrocellulose membrane with the total protein amount of, 16, 8, 4, 2 and 1ng. The membranes was dried in room temperature and stored in room temperature until use. Blot was blocked with TBS + 1% dry milk + 0.3% tween-20 for 1 hour at room temperature with agitation, Blot was incubated in the primary antibody at a dilution of 1: 1 000 in blocking solution ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation.Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:4000 in for 1h/RT with agitation. The blot was washed as above and developed for 2 min with chemiluminescent detection. Exposure time was 30 seconds.
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