Immunodetection of Amyloid Fibrils and Oligomers

Aliquots containing 100 ng Ca²⁺-bound monomers and amyloids of Gad m 1 chains were spotted in duplicate on a nitrocellulose membrane. Immunodetection was performed by 1 h of incubation with anti-amyloid fibril OC (AB2286 Merck Millipore, 1/2000 dilution) and anti-amyloid oligomer A11 (AB9234 Merck Millipore, 1/2000 dilution) antibodies, followed by extensive washes and 30 min of incubation with horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000 diluted; Sigma-Aldrich) [25 (link),26 (link)]. ECL Western blotting reagent (BioRad) and a ChemiDoc XRS instrument (BioRad) were used for signal development and detection, respectively.

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Castellanos M., Torres-Pardo A., Rodríguez-Pérez R, & Gasset M. (2018). Amyloid Assembly Endows Gad m 1 with Biomineralization Properties. Biomolecules, 8(1), 13.

Publication 2018

AB2286 AB9234 horseradish peroxidase-labeled goat anti-rabbit IgG ECL Western blotting reagent ChemiDoc XRS instrument

Amyloid Amyloid fibril Amyloids Anti igg Antibodies Dilution Goat Horseradish peroxidase Membrane Nitrocellulose Rabbit

Corresponding Organization : Consejo Superior de Investigaciones Científicas

Other organizations : IMDEA Nanoscience, Universidad Complutense de Madrid, Hospital Universitario La Paz

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Variable analysis

independent variables

Aliquots containing 100 ng Ca2+-bound monomers of Gad m 1 chains
Aliquots containing amyloids of Gad m 1 chains

dependent variables

Immunodetection results of anti-amyloid fibril OC (AB2286 Merck Millipore, 1/2000 dilution) and anti-amyloid oligomer A11 (AB9234 Merck Millipore, 1/2000 dilution) antibodies

control variables

Incubation time of 1 h with anti-amyloid fibril OC and anti-amyloid oligomer A11 antibodies
Extensive washes after antibody incubation
Incubation time of 30 min with horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000 diluted; Sigma-Aldrich)
Use of ECL Western blotting reagent (BioRad) for signal development
Use of ChemiDoc XRS instrument (BioRad) for signal detection

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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