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Sequential extraction and immunoblotting v1
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This protocol examines the fraction of alpha-synuclein (as assessed by alpha-synuclein and/or PS129 western blot) that is present in the triton-soluble or SDS-soluble fraction. Addition of alpha-synuclein pre-formed fibrils (PFFs) to neuron cultures seeds the recruitment of endogenous or transgenic alpha-synuclein into aggregates characterized by detergent insolubility. Monomeric alpha-synuclein will be present in the triton-soluble fraction, whereas PFF-induced alpha-synuclein oligomers will be present in the triton-insoluble, SDS-soluble fraction. This protocol encompasses preparation of cell extracts with Triton X-100, followed by sequential extraction of the Triton-insoluble material with SDS. The protein in the different detergent fractions are quantified by BCA, followed by SDS-PAGE and WB for alpha-synuclein, PS129, TUJ1, and loading control such as GAPDH.
ZappyLab, Inc.
Title: Sequential extraction and immunoblotting v1
Description:
This protocol examines the fraction of alpha-synuclein (as assessed by alpha-synuclein and/or PS129 western blot) that is present in the triton-soluble or SDS-soluble fraction.
Addition of alpha-synuclein pre-formed fibrils (PFFs) to neuron cultures seeds the recruitment of endogenous or transgenic alpha-synuclein into aggregates characterized by detergent insolubility.
Monomeric alpha-synuclein will be present in the triton-soluble fraction, whereas PFF-induced alpha-synuclein oligomers will be present in the triton-insoluble, SDS-soluble fraction.
This protocol encompasses preparation of cell extracts with Triton X-100, followed by sequential extraction of the Triton-insoluble material with SDS.
The protein in the different detergent fractions are quantified by BCA, followed by SDS-PAGE and WB for alpha-synuclein, PS129, TUJ1, and loading control such as GAPDH.
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