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.

Begin by placing a freshly dissected or snap-frozen brain tissue region in a two-milliliter tube containing six to eight ceramic beads and freshly prepared ice-cold homogenization buffer. Then, grind the tissue using a bead mill homogenizer at 4,000 RPM, with two cycles of 30 seconds on and off pulse. Now, add nine milliliters of ice-cold homogenization buffer to the one milliliter of brain tissue homogenate in a 15-milliliter tube, and seal with laboratory wax film strips.

To ensure robust solubilization, keep the tube rotating overnight at 4 degrees Celsius. The following day, add solid sucrose to the tissue extract suspension to a final concentration of 1.2 moles. Mix well and centrifuge at 250,000 times g for 45 minutes at 4 degrees Celsius. After discarding the supernatant, resuspend the pellet in two milliliters of homogenization buffer containing 1.9 molar sucrose by triturating and centrifuge at 125,000 times g for 45 minutes at 4 degrees Celsius.

After centrifugation, transfer the top white solid layer to a fresh tube, and solubilize in 1 milliliter of ice-cold wash buffer by pipetting up and down several times. Since the pellet is also enriched with amyloid fibrils, discard the aqueous middle layer, and combine the pellet with the top layer for a higher yield. Centrifuge the combined fractions at 8,000 times g for 20 minutes at 4 degrees Celsius.

After discarding the supernatant, resuspend the pellet in 1 milliliter of ice-cold digestion buffer, and incubate at room temperature for three hours on a vortex. Centrifuge the sample again, then, wash the pellet twice in 1 milliliter of ice-cold Tris buffer, and centrifuge again. After the second wash, resuspend the pellet in 1 milliliter of solubilization buffer by pipetting up and down, and quickly centrifuge the tube at 200,000 times g for 60 minutes at 4 degrees Celsius.

Save the pellet, then, reduce the sucrose concentration from 1.3 to 1 molar by adding 50 millimolar Tris buffer to the supernatant. Centrifuge the supernatant again, then, dissolve both pellets in 100 microliters of Tris buffer containing 0.5% SDS. For amyloid purification, solubilize the amyloid-rich pellets using ultrasound waves in a bath sonication device for 20 cycles. Then, immediately centrifuge the material at 20,000 times g for 30 minutes at 4 degrees Celsius.

Resuspend the pellet in 500 microliters of 0.5% SDS Tris buffer, and repeat the washing four more times. After the final centrifugation step, wash the pellet in 200 microliters of ultrapure water, and centrifuge at 20,000 times g for 30 minutes at 4 degrees Celsius to remove any remaining detergent. Dissolve the final pellet containing purified amyloid fibrils in 100 microliters of ultrapure water.