Isolation of Amyloid Fibrils from Brain Tissue Extract

Take a mouse brain-tissue extract containing non-amyloid proteins and amyloid fibrils, an insoluble protein aggregate.

Add solid sucrose and mix, increasing the solution's density.

Transfer it and centrifuge to separate denser proteins from debris.

Discard the supernatant, resuspend the pellet in a higher sucrose concentration buffer, mix and centrifuge.

Collect the top layer containing a few amyloid fibrils in a fresh tube, add a wash buffer, and mix.

Now, discard the middle layer and transfer the amyloid fibrils-enriched pellet to the tube containing the top layer fraction for maximum isolation.

Centrifuge the combined fractions and discard the supernatant. Add a digestion buffer and incubate to degrade non-amyloid proteins exclusively.

Centrifuge and remove the supernatant. Wash the pellet to remove residual digested proteins.

Resuspend the pellet in a sucrose-based solubilization buffer containing detergent that solubilizes the remaining non-amyloid proteins.

Centrifuge, remove the supernatant, and resuspend the isolated amyloid fibrils.