鼠脑组织匀浆中的Aβ蛋白的提取

Brad T. Casali; Gary E. Landreth

doi:10.21769/BioProtoc.1787

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Peer-reviewed

Aβ Extraction from Murine Brain Homogenates

鼠脑组织匀浆中的Aβ蛋白的提取

BC Brad T. Casali

GL Gary E. Landreth email

发布: 2016年04月20日第6卷第8期 DOI: 10.21769/BioProtoc.1787 浏览次数: 11448

评审: Oneil G. BhalalaGeoff LauKae-Jiun Chang

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Jun 2015

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Abstract

This protocol details beta-amyloid (Aβ) extraction from transgenic murine brain homogenates. Specifically, mechanical homogenization of brain tissue and sequential extraction of both soluble and insoluble proteins are detailed. DEA extracts soluble proteins, such as Aβ isoforms and APP. Formic acid enables extraction of insoluble protein aggregates, such as Aβ isoforms associated with plaques. This procedure produces soluble and insoluble extracts that are amenable to analysis of Aβ species via western blotting and/or enzyme-linked immunosorbent assays (ELISAs), and these results help assess amyloidogenic burden in animals.

Keywords: Beta-amyloid (淀粉样蛋白)

Materials and Reagents

5.0 ml open-top polyallomer ultracentrifuge tubes (or tubes capable of undergoing high-speed centrifugation) (Denville Scientific, catalog number: U5022 )
Diethylamine (DEA) (≥ 99.5%) (Sigma-Aldrich, catalog number: 471216 )
95% formic acid (FA) (AMRESCO, catalog number: 0961 )
100 mM NaCl (store at room temperature)
Tris base (Thermo Fisher Scientific, catalog numeber: BP152 )
0.5 M sodium phosphate dibasic (Na₂HPO₄) (AMRESCO, catalog numeber: 0348 )
0.05% sodium azide (NaN₃) (Thermo Fisher Scientific, catalog numeber: S2271 )
250 mM sucrose (Thermo Fisher Scientific, catalog numeber: S5 )
0.5 mM Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate (EDTA) (Thermo Fisher Scientific, catalog numeber: S311 )
0.5 mM Ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) (Sigma Aldrich, catalog numeber: 03777 )
Tris-hydrochloride (Tris-HCl)
0.4% DEA in 100 mM NaCl (see Recipes)
0.5 M Tris-HCl (pH 6.8) (see Recipes)
Formic acid neutralization buffer (see Recipes)
Tissue homogenization buffer (THB) (see Recipes)
Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 ) (see Recipes)

Equipment

Beckman Coulter Optima L-90K Ultracentrifuge (used with an SW50.1 rotor)
Ultrasonic sonicator (see Note 7, below) (Kontes, model: KT50 , catalog number: 12038 )

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Casali, B. T. and Landreth, G. E. (2016). Aβ Extraction from Murine Brain Homogenates. Bio-protocol 6(8): e1787. DOI: 10.21769/BioProtoc.1787.
Casali, B. T., Corona, A. W., Mariani, M. M., Karlo, J. C., Ghosal, K. and Landreth, G. E. (2015). Omega-3 fatty acids augment the actions of nuclear receptor agonists in a mouse model of Alzheimer's disease. J Neurosci 35(24): 9173-9181.