Detecting Neurodegenerative Disease-Associated Protein α-Synuclein Using ELISA

Begin with the brain tissue from a transgenic mouse overexpressing mutated α-synuclein, a misfolded neuronal protein associated with neurodegenerative disease, immersed in a high-salt buffer.

Homogenize the tissue with a large pestle, followed by a small pestle to disrupt the tissue and release intracellular proteins, including α-synuclein.

Transfer the homogenate to a tube and centrifuge. Collect the protein-containing supernatant and dilute it with a buffer.

Add this supernatant to an α-synuclein-specific antibodies-coated microplate pretreated with a blocking agent to prevent nonspecific binding.

Incubate with agitation, allowing normal and mutated α-synuclein to bind.

Wash the plate, then add detection antibodies specific to mutated α-synuclein and incubate with agitation.

Wash again and add enzyme-linked antibodies, followed by incubation under agitation.

Add substrate and incubate in the dark under agitation. The substrate reacts with the enzyme to produce a color change.

Stop the reaction with an acid and measure the absorbance to quantify mutated α-synuclein.

To prepare brain homogenates, add an adequate volume of HS buffer to the dissected brain regions to reach an expected percen