Non-denaturing Immuno-Dot Blot for Alpha-Synuclein

Samples were spotted by non-denaturing immuno-dot blot as previously described [53 (link)]. Briefly, different amounts of PFF (3.12–100 ng), monomeric α-Syn (100 ng), or vehicle (PBS) were dotted directly onto a nitrocellulose membrane using a vacuum filtration system (Bio-Rad Bio-Dot Apparatus). Membranes were incubated with primary antibodies anti-α-Syn Syn-1 (BD Biosciences #610,787, 1:1000), anti-p25α (produced in house [58 (link)], affinity rabbit polyclonal, 1:1000), and aggregate-specific MJF14-6–4-2 (MJF14) (Abcam #209,538, 1:45,000) and fibril-specific FILA-1 (produced in house [59 (link)], 1:1,000). Proteins were visualised using ECL in a Fuji LAS-3000 Intelligent Dark Box (Fujifilm, Japan).

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Ferreira N., Gram H., Sorrentino Z.A., Gregersen E., Schmidt S.I., Reimer L., Betzer C., Perez-Gozalbo C., Beltoja M., Nagaraj M., Wang J., Nowak J.S., Dong M., Willén K., Cholak E., Bjerregaard-Andersen K., Mendez N., Rabadia P., Shahnawaz M., Soto C., Otzen D.E., Akbey Ü., Meyer M., Giasson B.I., Romero-Ramos M, & Jensen P.H. (2021). Multiple system atrophy-associated oligodendroglial protein p25α stimulates formation of novel α-synuclein strain with enhanced neurodegenerative potential. Acta Neuropathologica, 142(1), 87-115.

Publication 2021

Bio-Dot Apparatus anti-α-Syn Syn-1 Fuji LAS-3000 Intelligent Dark Box

Anti antibodies Dot blot Filtration Membranes Nitrocellulose Proteins Rabbit Syn 1 Vacuum

Corresponding Organization : Aarhus University

Other organizations : University of Florida, University of Southern Denmark, Lundbeck (Denmark)

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Variable analysis

independent variables

Amount of PFF (3.12–100 ng)
Amount of monomeric α-Syn (100 ng)
Vehicle (PBS)

dependent variables

Binding of primary antibodies (anti-α-Syn Syn-1, anti-p25α, aggregate-specific MJF14, fibril-specific FILA-1) to the samples
Protein visualization using ECL

control variables

Nitrocellulose membrane
Vacuum filtration system (Bio-Rad Bio-Dot Apparatus)
Incubation conditions for primary antibodies
ECL detection method

positive controls

Monomeric α-Syn (100 ng)

negative controls

Vehicle (PBS)

Annotations

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