Isolation of Amyloid Fibrils from Brain Tissue Extract

Abstract

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....

Results

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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