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, ELISA, Extraction, Abeta, Murine
Materials and Reagents
Equipment
Procedure
Note: The following protocol has been used to extract Aβ from multiple mouse models of Alzheimer’s disease [please see Cramer et al. (2012) and Casali et al. (2015)]. The user may need to modify dilutions of the final extracted product depending on the particular application (e.g., ELISA and/or Western blotting). Our lab usually dilutes DEA and FA fractions for Aβ ELISAs at least five-fold to fall within our in-house ELISA detection limits. For Western blots of Aβ and modified APP fragments, we recommend 10 to 50 micrograms protein per well, and for more details about Western blotting using the DEA-soluble extraction, please see Morales-Corraliza et al. (2012).
Notes
Recipes
Acknowledgments
This work was supported by a grant from the National Institutes of Health, R41-AG048658.
References
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