Isolation of Detergent-Insoluble Protein Aggregates From Human Postmortem Brain Tissue

Begin with a frozen postmortem human brain tissue sample from a confirmed neurodegenerative disease case.

Excise grey matter rich in insoluble protein aggregates, a hallmark of the disease.

Transfer the tissue to a homogenizer.

Add a buffer containing inhibitors and homogenize to release intracellular components.

The inhibitors block protease and phosphatase enzymes, preventing protein degradation.

Transfer the homogenate to a tube and add an ionic detergent buffer. Incubate.

The detergent solubilizes natively folded proteins, while misfolded protein aggregates remain intact.

Sonicate to complete cellular dissociation.

Transfer the homogenate to an ultracentrifuge tube and centrifuge at high speed.

The aggregates settle at the bottom.

Remove the supernatant containing detergent-soluble proteins.

Wash the aggregates with an ionic detergent buffer, centrifuge, and remove the supernatant.

Add urea buffer to denature the protein aggregates, making them soluble.

Transfer the solubilized protein aggregates to a tube and sonicate briefly to ensure complete dissolution.