Detection of Phosphorylated Alpha-Synuclein Using Western Blotting

Begin with CSF proteins, including phosphorylated alpha-synuclein, a biomarker of neurodegenerative diseases.

Add a loading buffer containing a detergent, a reducing agent, and a tracking dye.

Heat to denature the proteins, then load them into an SDS-PAGE gel.

Perform electrophoresis to separate the proteins by molecular weight into distinct bands.

Incubate the gel in a blotting buffer with agitation.

Then, electro-transfer the protein bands from the gel onto the membrane.

Treat the membrane with a fixative to retain the protein on the membrane, then wash.

Incubate with a blocking solution containing primary antibodies and phosphatase inhibitors to maintain alpha-synuclein's phosphorylation.

The antibodies exclusively interact with phosphorylated alpha-synuclein, and the blocking reagents block nonspecific interaction sites, then wash.

Introduce enzyme-conjugated secondary antibodies that interact with primary antibodies, then wash.

Add a chemiluminescent substrate, interacting with enzymes and generating a chemiluminescence.

A chemiluminescent band confirms the presence of phosphorylated alpha-synuclein.