Feb 28, 2024
ELISA (enzyme-linked immunosorbent assay) for α-Syn quantification
- 1University of Ottawa
Protocol Citation: Jean-Louis Parmasad 2024. ELISA (enzyme-linked immunosorbent assay) for α-Syn quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.261gedywyv47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
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Created: February 28, 2024
Last Modified: February 28, 2024
Protocol Integer ID: 95878
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Abstract
ELISA (enzyme-linked immunosorbent assay) for α-Syn quantification. Useful for analyses of alpha-Synuclein and related proteins in the context of synucleinopathy.
Coat 384-well MaxiSorp plates (Nunc, Inc) with capturing antibody (α-Syn, BD Biosciences, 610787) diluted 1:500 in coating buffer (NaHCO3 with 0.2% NaN3, pH9.6) overnight at 4 °C.
Wash 3 times with PBS.0.05% Tween-20 (PBS-T).
Block for 1hr at 37 °C in blocking buffer (1.125% fish skin gelatin; PBS-T)
Load samples in duplicate into wells.
Incubate at RT for 2hr.
Add the desired antibody to the plate for 1 h at 37 °C
Wash 5 times with PBS-T.
Add ExtrAvidin phosphatase (Sigma, E2636) diluted in blocking buffer to wells. Incubate for 30 min at 37 °C.
Carry out colour development with fast-p-nitrophenyl phosphate (Sigma, N1891) at 405 nm every 2.5 min for up to 60 min.
Identify time point(s) where standards and sample dilutions are in the log phase.