Isolation of Amyloid Fibrils from Brain Tissue Extract

Samenvatting

Source: Upadhyay, A. et al., Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain. J. Vis. Exp. (2022)

This video demonstrates the protocol for isolating amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Tissue harvesting and amyloid purification

NOTE: Ideally, amyloid fibrils should be isolated from freshly dissected brain regions. However, this method also works well with snap- or flash-frozen brain tissues. Below is a brief outline of snap-freezing brain tissues for storage for use at a later time.

1. Brain tissue harvesting and storage: Dissect mouse brains and quickly harvest the amyloid-laden regions (i.e., cortex and hippocampus), followed by snap freezing in liquid nitrogen or a dry ice alcohol bath.
   NOTE: Snap freezing helps to preserve the structural attributes of the tissue's constituents. The mice are euthanized by isoflurane and cervical dislocation. It is advised to avoid using CO₂ since that can compromise brain proteome integrity.
2. Post-dissection, cut, and store fresh tissues in small blocks (e.g., 5 mm x 5 mm), as this facilitates more efficient homogenization before amyloid extraction. Transfer the tissue to the sterile cryogenic vial, tighten its cap, and rapidly submerge.
3. Keep tissues frozen in ultracold conditions (i.e., -80 °C or liquid nitrogen). If extraction is to be performed a few days later, store the tissues at -20 °C and avoid freeze and thaw cycles. Thaw the frozen tissues on wet ice when ready for extraction and purification.
   ​NOTE: Direct contact of the tissues with liquid nitrogen can cause tissue and protein damage. Note that an alcohol bath can remove permanent markers from the tube labels.

2. Enrichment of sodium dodecyl sulphate (SDS) insoluble material

NOTE: Perform all the steps on ice and centrifuge at 4 °C, unless stated otherwise. Manufacturers and catalog numbers of chemicals and instruments are provided in the Table of Materials.

1. Start with freshly dissected or snap-frozen brain tissue regions (thawed on ice) placed into a 2 mL tube containing 6-8 ceramic beads and freshly prepared ice-cold homogenization buffer (1 mL for 0.25-1 g of wet tissue mass).
2. Transfer the tubes to the bead mill homogenizer and grind the tissue contents at 4000 rpm, with two cycles of 30 s each and an interval of 30 s in between.
   NOTE: Alternatively, motorized stirrer or homogenizer systems can be used to grind and homogenize the tissues.
3. Add 9 mL of ice-cold homogenization buffer to the 1 mL of brain whole tissue homogenate in 15 mL tubes, seal with laboratory wax film strips, and rotate end-to-end overnight at 4 °C to ensure robust solubilization.
   NOTE: To remove unwanted large cellular debris and lipids, on the next morning, spin the tubes at 800 x g at 4 °C for 10 min and collect the supernatant in a fresh tube. Resuspend the remaining pellet in 2 mL of ice-cold homogenization buffer, mix well, spin again for 10 min at 4 °C, and combine the two supernatants.
4. Slowly add solid sucrose to the tissue extract suspension to a final concentration of 1.2 M, mix well, and centrifuge at 250,000 x g for 45 min at 4 °C.
5. Carefully remove and discard the supernatant using a pipette. Resuspend the pellet in 2 mL of homogenization buffer containing 1.9 M sucrose by triturating, followed by centrifugation at 125,000 x g for 45 min at 4 °C.
   NOTE: The buffer volume can be adjusted based on the amount of material recovered from the previous step. In general, 10 volumes of homogenization buffer (V/V) is ideal for this step.
6. Collect the top white solid layer using a pipette, transfer it to a fresh tube, and solubilize in 1 mL of ice-cold wash buffer by pipetting up and down several times, without introducing air bubbles.
7. Apart from the top layer, the pellet is also enriched with amyloid fibrils. For a higher yield, combine the two fractions and proceed. Carefully remove the middle aqueous layer using a pipette and discard.
8. Centrifuge the combined fractions at 8000 x g for 20 min at 4 °C. Discard the supernatant.
9. Add 1 mL of ice-cold digestion buffer to the washed pellet, resuspend, and incubate at room temperature (RT) for 3 h.
10. Centrifuge at 8000 x g for 20 min at 4 °C and remove the supernatant using a pipette.
11. Resuspend and wash (same as Step 2.8.) the digested pellet two times in 1 mL of ice-cold Tris buffer.
12. Resuspend the washed pellet in 1 mL of solubilization buffer containing 1% SDS and 1.3 M sucrose by pipetting up and down. Centrifuge quickly at 200,000 x g for 60 min at 4 °C.
    NOTE: Although the amyloid fibrils are highly resistant to detergents and denaturants, very long exposures to the detergent (1% SDS) may affect the integrity of the fibrils or remove the tightly bound proteins, which will compromise subsequent analyses. Therefore, immediately after resuspending the pellets, proceed to centrifugation.
13. Save the pellet and increase the volume of the remaining supernatant by adding 50 mM Tris buffer (in ratio 1:0.3) to reduce the sucrose concentration from 1.3 to 1 M and centrifuge one more time at 200,000 x g for 45 min at 4 °C.
14. Dissolve the two pellets in 100 µL of Tris buffer containing 0.5% SDS and pool for amyloid purification. Visible pellets are small and should appear opaque and off-white (see Figure 1A).

Resultaten

Figure 1: Confirmation of amyloid extraction using biochemical staining and imaging of amyloid fibrils. (A) Enriched amyloid-containing SDS insoluble pellet appears opaque off-white in color. (B) Congo red stain...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Protease Inhibito	Thermo Scientific	164535	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Calcium Chloride	Sigma-Aldrich	C1016	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Collagenase	Sigma-Aldrich	C0130	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Complete Protease Inhibitor Cocktail	Sigma-Aldrich	11697498001	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Dnase I	Thermo Fisher Scientific	EN0521	Alternative instruments, chemicals and antibodies from other manufacturers can be used
K54 Tissue Homogenizing System Motor	Cole Parmer	Glas-Col 099C	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Optima L-90K Ultracentrifuge	Beckman Coulter	BR-8101P-E	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Precellys 24 tissue homogenizer	Bertin Instruments	P000062-PEVO0-A	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	74255	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Sorvall Legend Micro 21R Microcentrifuge	Thermo Fisher Scientific	75002446	Alternative instruments, chemicals and antibodies from other manufacturers can be used
Tris-HCl	Thermo Fisher Scientific	15568025	Alternative instruments, chemicals and antibodies from other manufacturers can be used