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

Abstract

Source: Diner, I., et al. Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain. J. Vis. Exp. (2017).

This video explains the process of isolating and enriching detergent-insoluble protein aggregates from frozen postmortem human brain tissue. It demonstrates key steps, including homogenization, detergent treatment, sonication, and ultracentrifugation to separate and purify the insoluble aggregates. The final step involves solubilizing the aggregates in urea buffer for further analysis.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.  

1. Homogenization and Fractionation

1. Tissue selection
   NOTE: Frozen postmortem frontal cortex tissue from healthy control (Ctl) and pathologically confirmed AD cases were selected (n=2). Post-mortem neuropathological evaluation of amyloid plaque distribution was performed according to the Consortium to Establish a Registry for Alzheimer's Disease semi-quantitative scoring criteria, although neurofibrillary tangle pathology was assessed in accordance with the Braak staging system.
   1. Obtain frozen postmortem brain tissue from healthy control (Ctl) and pathologically confirmed AD cases. Utilizing containers of dry ice, forceps and a razor blade, excise ~ 250 mg portions of grey matter from each tissue sample on tared disposable weigh boats, taking care to prevent the tissue from thawing. Record the weight of each piece to be homogenized.
   2. Excise ~ 250 mg of grey matter using forceps and razor by visually inspecting to locate the interface between the outer grey matter and inner white matter. Avoid white matter and more importantly, any large blood vessels or bloody regions, meninges, or arachnoid mater. Keep the tissue frozen during the cutting process by working quickly and frequently placing weighing boat with brain tissue into polystyrene containers with dry ice.
   3. Record the exact weight of grey matter excised from each frozen piece using an analytical balance. Calculate the volume of low salt (LS) buffer (see Table 1) needed for dounce homogenization (5 mL/g or 20% w/v).
   4. Prepare 10 mL LS buffer with 1x protease and phosphatase inhibitor cocktail and chill on ice.
   5. Remove weighed tissue and weighing boat from dry ice and dice into roughly 2 x 2 mm pieces as it thaws. Transfer to a 2 mL pre-chilled dounce homogenizer tube on ice.
   6. Add 5 mL/g (20% w/v) of ice-cold low salt (LS) buffer with 1X protease and phosphatase inhibitor cocktail.
   7. Homogenize the brain tissue on ice using approximately 10 strokes of high clearance pestle A and 15 strokes of low clearance pestle B.
   8. Transfer all homogenate to a 2 mL polypropylene tube using a glass Pasteur pipette. Label the tube with "TH" (Total Homogenate), the tissue case number, and homogenate volume.  Aliquot 0.8 mL into a labeled 1.5 mL tube. Any excess homogenate can be similarly aliquoted and stored at -80 °C.
   9. To each 0.8 mL aliquot, add 100 µL each of 5 M NaCl and 10% (w/v) sarkosyl to concentrations of 0.5 M and 1% w/v, respectively. Mix tubes well by inversion and incubate on ice for 15 min. Label tubes with "TH-S" (Total Homogenate-Sarkosyl) to indicate that the homogenates are in sarkosyl-buffer (Table 1).
   10. Sonicate each tube for three 5 s pulses at 30% amplitude on ice (maximum intensity = 40%) using a microtip probe.
   11. Determine the protein concentrations of the homogenates using the bicinchoninic acid (BCA) assay method.
       NOTE: The average protein concentration of TH-S homogenates prepared at 5 mL LS per gram of tissue is 15-20 mg/mL. The homogenates can be fractionated immediately or stored at -80 °C until use.
   12. Dilute TH-S homogenates to 10 mg/mL using ice-cold sark buffer (Table 1) with 1x protease and phosphatase inhibitors.
   13. Transfer 5 mg protein (0.5 mL) of each TH-S homogenate into 500 µL polycarbonate ultracentrifuge tubes and pair-balance with sark-buffer. Load tubes into a pre-chilled rotor and ultracentrifuge at 180,000 x g for 30 min at 4 °C. Transfer the sarkosyl-soluble supernatants (S1) to 1.5 mL tubes and store at -80 °C.
       NOTE: (Optional wash) Add 200 µL of sark-buffer to the ultracentrifuge tubes containing the detergent-insoluble fractions (P1) and dislodge the pellets (P1) from the bottom of the ultracentrifuge tubes using a 200 µL pipette tip.
   14. Briefly pulse-spin the inverted tubes for 2-3 s (≤2,500 x g) on a microcentrifuge to transfer the pellets (P1) and buffer to the 1.5 mL tubes below. Resuspend the insoluble pellets (P1) in the sark-buffer by pipetting up and down to ensure the pellet is disrupted.
   15. Pair-balance, and centrifuge at 180,000 x g for an additional 30 min at 4 °C.
   16. Discard the optional wash supernatant (S2) and incubate the sarkosyl-insoluble pellets (P2) in 50-75 µL of urea buffer (Table 1) with 1x PIC (protease and phosphatase inhibitor cocktail) for 30 min at room temperature to solubilize the pellet.
       NOTE: Warm urea buffer to room temperature before use to avoid SDS precipitation. For detergent-sensitive applications, omit SDS from urea buffer and consider washing the insoluble pellet (P2) in low salt buffer before resuspending in urea buffer.
   17. Transfer the resuspended pellets (P2) to 0.5 mL tubes and use brief (1 s) microtip sonication at 20% amplitude (maximum intensity = 40%) to fully solubilize the pellets.
   18. Determine the protein concentrations of the sarkosyl-soluble (S1) and -insoluble (P2) fractions using the BCA assay method. Use these fractions immediately or store at -80 °C until use.

Table 1: Buffers list

10% w/v sarkosyl solution: 10 g N-lauryl-sarcosine sodium salt per 100 ml of ddH2O (stir at RT overnight)

Low Salt (LS) buffer: 50 mM HEPES pH 7.0, 250 mM sucrose, 1 mM EDTA

Sarkosyl (sark) buffer: LS buffer + 1% (w/v) sarkosyl + 0.5 M NaCl

Urea buffer (store at -20 °C): 50 mM Tris-HCl pH 8.5, 8M urea, 2% SDS

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protease and phosphatase inhibitor cocktail, EDTA-free (100X)	Thermo Fisher	78441	protease & phosphatase inhibitor cocktail
Sonic Dismembrator System (ultrasonicator)	Fisher Scientific	FB505110	microtip ultrasonicator
Optimax TLX Ultracentrifuge	Beckman Coulter	361545	refrigerated ultracentrifuge
TLA120.1 rotor	Beckman Coulter	362224	ultracentrifuge rotor
500 ul (8x34mm) polycarbonate tubes, thickwall	Beckman Coulter	343776	ultracentrifuge tubes for TLA120.1 rotor
N-Lauroylsarcosine sodium salt (sarkosyl)	Sigma Aldrich	L5777-50G	detergent
1.5 ml polypropylene Pellet Pestle	Kimble Chase	749521-1500	homogenization tool
Cordless motor for Pellet Pestle	Kimble Chase	749540-0000	homogenization tool