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Cited by 403Alzheimer's disease research and treatmentsProtein purification and stabilityDementia and Cognitive Impairment Research

Preparing Synthetic Aβ in Different Aggregation States

Open access
Stine, W. Blaine, Jungbauer, Lisa, Yu, Chunjiang, LaDu, Mary Jo·2010·Methods in Molecular Biology
DOI 10.1007/978-1-60761-744-0_2PMC PMC3752843
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Abstract

This chapter outlines protocols that produce homogenous preparations of oligomeric and fibrillar amyloid-β peptide (Aβ). While there are several isoforms of this peptide, the 42 amino acid form is the focus because of its genetic and pathological link to Alzheimer's disease (AD). Past decades of AD research highlight the dependence of Aβ42 function on its structural assembly state. Biochemical, cellular and in vivo studies of Aβ42 usually begin with purified peptide obtained by chemical synthesis or recombinant expression. The initial steps to solubilize and prepare these purified dry peptide stocks are critical to controlling the structural assembly of Aβ. To develop homogenous Aβ42 assemblies, we initially monomerize the peptide, erasing any "structural history" that could seed aggregation, by using a strong solvent. It is this starting material that has allowed us to define and optimize conditions that consistently produce homogenous solutions of soluble oligomeric and fibrillar Aβ42 assemblies. These preparations have been developed and characterized by using atomic force microscopy (AFM) to identify the structurally discrete species formed by Aβ42 under specific solution conditions. These preparations have been used extensively to demonstrate a variety of functional differences between oligomeric and fibrillar Aβ42. We also present a protocol for fluorescently labeling oligomeric Aβ42 that does not affect structure, as measured by AFM, or function, as measured by a cellular uptake assay. These reagents are critical experimental tools that allow for defining specific structure/function connections.

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  • The Alamar Blue cell assay is more appropriate for Aβ toxicity studies than the MTT assay.

    “The viability of Neuro-2A (N2A) cells57 was probed using the Alamar Blue cell assay, which was shown to be more appropriate for Aβ toxicity studies vs. the MTT assay.58,59”

    From The effect of Cu2+ and Zn2+ on the Aβ42 peptide aggregation and cellular toxicity (2013) · cites this paper as [59]

    The Materials (§2.8.1) and Methods (§3.8.1) describe an Aβ toxicity assay in N2A cells using CellTiter-Glo to measure cellular ATP. Searches for Alamar Blue and MTT found no passage comparing those assays or saying Alamar Blue is more appropriate, so our copy does not verify the attributed comparison. (LLM, conf=0.75)

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  • The Alamar Blue cell viability assay has been shown to give more reproducible results than the MTT assay.

    “using an Alamar Blue cell viability assay, which has been shown to give more reproducible results than the MTT assay.93,94”

    From Bifunctional Compounds for Controlling Metal-Mediated Aggregation of the Aβ<sub>42</sub>Peptide (2012) · cites this paper as [94]

    The Methods section (3.8.1, page not available) describes a CellTiter-Glo ATP-based viability assay, not an Alamar Blue assay. Searches for Alamar Blue, MTT, and assay reproducibility found no comparison between them in our copy. (LLM, conf=0.76)

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Cites

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Research works printed but not linked: 26

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Authors
Stine, W. B., Jungbauer, L., Yu, C., & LaDu, M. J.
Affiliations
University of Illinois Chicago (US)
ORCID
Venue
Methods in Molecular Biology
Publisher
Humana Press
Published
2010
Volume
670
Pages
13-32
ISSN
  • 1064-3745 (ISSN-L)
  • 1940-6029
DOI
10.1007/978-1-60761-744-0_2
PMC
PMC3752843
OpenAlex
W1780440403
Cited by
403
References
6
Citations by year
2026: 28 · 2025: 43 · 2024: 41 · 2023: 38 · 2022: 48 · 2021: 41 · 2020: 35 · 2019: 24 · 2018: 23 · 2017: 27 · 2016: 15 · 2015: 10 · 2014: 8 · 2013: 9 · 2012: 5
FWCI
22.89
Type
book chapter
Language
en
Open access
Locations
  • Methods in molecular biology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health (5f32ag030256-02)
  • National Institute on Aging (P01AG030128, R01AG019121, 1F32 AG030256-01, R01 AG19121, P01AG030128-A2, F32 AG030256, P01AG021184)
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
Keywords
amyloid-beta 42, peptide aggregation, amyloid-beta oligomers, fibrillar amyloid-beta, Alzheimer's disease, peptide solubilization, monomerization, fluorescent labeling, structure-function relationship, atomic force microscopy
MeSH terms
Animals, Cell Survival, Electrophoresis, Polyacrylamide Gel, Blotting, Western, Amyloid beta-Peptides, Microscopy, Atomic Force, Cell Line, Tumor, Mice

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