SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

Summary

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology.

Abstract

The anomalous folding and polymerization of the β-amyloid (Aβ) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis¹. Aβ is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into β-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble Aβ multimers are more toxic than fibrillar Aβ amyloid². The identification and quantification of low- and high-molecular weight multimeric Aβ species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies³. Naturally occurring Aβ multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Aβ-specific antibodies. However, the separation and detection of multimeric Aβ requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the Aβ peptide. Using this protocol, we consistently detect Aβ monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

Protocol

Part 1: Preparation of clarified tissue homogenates

1. Dounce homogenize unfixed cortical tissue in 4 times volume of ice-cold buffer (0.1M phosphate-buffered saline [Ca++- and Mg++-free] plus 2x protease inhibitor cocktail [Santa Cruz]) with approximately 30 even pestle strokes (i.e. add 400μl buffer to 100mg tissue).
2. Spin homogenates for 5 minutes at 3,000g (4°C). Carefully remove clarified supernatants and store aliquots of these extracts at -80°C until use.
3. Determine total protein concentration (μg/μl) for clarified homogenates using a bicinchoninic acid (BCA) assay, according to the manufacturer's instructions (ThermoFishe....

Discussion

Despite the importance of Aβ aggregation in the pathogenesis of Alzheimer's disease^1,4,5 , few studies have described or quantified the distribution of diverse Aβ multimers in human cortical tissue samples². Commonly used immunohistochemical techniques do not allow for the discrimination of distinct multimeric Aβ species in fixed cortical tissue. In unfixed cortical tissue homogenates, Aβ multimers can be separated and biochemically assessed using gel electrophoresis and antibod.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Complete Protease Inhibitor Cocktail Tablets	Santa Cruz Biotechnology, Inc.	Sc-29130	1 tablet in 25ml buffer
BCA Protein Assay kit	Thermo Fisher Scientific, Inc.	23225
XCell SureLock Mini-Cell and XCell II Blot Module Kit CE Mark	Invitrogen	EI0002
Novex Tricine SDS Sample Buffer (2X)	Invitrogen	LC1676
NuPAGE Sample Reducing Agent (10X)	Invitrogen	NP0004
SeeBlue Plus2 Pre-Stained Standard	Invitrogen	LC5925
Novex 10-20% Tricine Gel 1.0 mm, 10 well	Invitrogen	EC6625BOX
Nitrocellulose membranes, 0.2 μm pore size	Invitrogen	LC2000
Novex Tricine SDS Running Buffer (10X)	Invitrogen	LC1675
Novex Tris-Glycine Transfer Buffer (25X)	Invitrogen	LC3675
SimplyBlue SafeStain	Invitrogen	LC6060	Will not interfere with immunostaining
ATX Ponceau S Red staining solution	Sigma-Aldrich	09276	Will not interfere with immunostaining
Kapak heat sealable plastic sample pouches	Fisher Scientific	0181225AA
6E10 mouse monoclonal antibody to Aβ(1-16)	Covance	SIG-39320	Dilute 1:1,000 up to 1:5,000 for WB
Tween 20	Sigma-Aldrich	P2287
ECL Mouse IgG, HRP-Linked Whole Aβ (from sheep)	GE Healthcare	NA931-1ML	Dilute at 1:10,000
SuperSignal West Pico Chemiluminescent Substrate	Thermo Fisher Scientific, Inc.	34077
Kodak Biomax MR Film	Carestream Health	870 1302