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....

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