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.  

Obtain frozen postmortem brain tissue from healthy control and pathologically confirmed AD. Use forceps and a razor blade to excise approximately 250 milligram portions of gray matter from each tissue sample. Allow the brain segment to thaw for one minute. Now, dice a piece of tissue into roughly two-millimeter cubed pieces as it thaws. Transfer to a two-milliliter pre-chilled dounce homogenizer tube on ice.

Remember to avoid white matter and any large blood vessels.

Work quickly and place the tissue on tear disposable weigh boats. Take care to prevent the tissue from thawing by frequently placing the weighing boat with brain tissue into polystyrene containers with dry ice. After dissecting and weighing all required pieces of tissue, remove a weigh boat containing a piece of tissue from dry ice.

Next, add five milliliters of ice cold low-salt buffer with protease and phosphatase inhibitor cocktail per gram of tissue, and homogenize on ice using approximately 10 strokes of high-clearance pestle A and 15 strokes of low-clearance pestle B. Then, use a nine-inch glass Pasteur pipette to transfer each homogenate to a labeled two-milliliter polypropylene tube. Aliquot the remaining homogenate into additional labeled 1.5-milliliter tubes in a similar fashion for storage at -80 degrees Celsius.

To proceed with fractionation, add 100 microliters of five molar sodium chloride and 100 microliters of 10% Sarkosyl to each 0.8-millimeter aliquot. Mix well by inversion, and then, incubate on ice for 15 minutes. Following the incubation, use a sonicator equipped with a micro-tip probe to deliver three 5-second pulses at 30% amplitude.

After using the bicinchoninic acid method to determine the protein concentrations of the homogenates, add ice-cold sark buffer with protease and phosphatase inhibitors to each sample to a final concentration of 10 milligrams per millimeter. Then, transfer 500 microliters of each sample into 500 microliter polycarbonate ultracentrifuge tubes.

Load tubes into a pre-chilled rotor, and ultracentrifuge at 180,000 times g for 30 minutes at 4 degrees Celsius. Following the centrifugation, transfer the S1 sarkosyl soluble supernatants to 1.5-milliliter tubes and store at -80 degrees Celsius. Add 200 microliters of sark buffer to the ultracentrifuge tubes containing the detergent insoluble P1 fractions and use the pipette tip to dislodge the pellets from the bottom of the ultracentrifuge tubes.

Next, transfer the resuspended P1 pellets to new 500-microliter ultracentrifuge tubes, pair balance, and centrifuge at 180,000 times g for an additional 30 minutes at 4 degrees Celsius. Discard the S2 supernatant, then add 50 to 75 microliters of urea buffer with protease and phosphatase inhibitor cocktail to the sarkosyl insoluble pellets and incubate for 30 minutes at room temperature to solubilize the P2 pellet.

After the incubation, transfer the resuspended P2 pellets to labeled 0.5-millimeter tubes and fully solubilize the pellets using a brief 1 second microtip sonication at 20% amplitude. Determine the protein concentrations of the sarkosyl soluble and sarkosyl insoluble fractions using the BCA assay method.