Abstract
The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like Western-blot or ELISA in e.g., mouse models of Alzheimer’s disease or other neurodegenerative conditions.
Keywords: Brain, Synaptosome, Fractionation, Homogenization, Extraction, Synapse
Background
Analyzing synaptosomes, representing isolated synaptic terminals from neurons, can yield valuable information on synaptic integrity in diverse neurological diseases. They contain membrane-bound compartments that detach from axon terminals after brain homogenization under certain conditions. The current protocol describes a fast and easy method for the enrichment of crude synaptosomal fractions (see Figure 1). These can be either used for quantification of synaptic proteins by Western-blot or can be further purified using density gradient centrifugation to yield highly purified synaptosome subfractions (Gurd et al., 1974). The preparation of crude synaptosomal fractions might be sufficient to assess e.g., the amount of pre- and post-synaptic proteins like SNAP25 or post-synaptic density protein 95 (PSD95) in e.g., mouse models with a neurodegenerative phenotype (Breyhan et al., 2009; Saul and Wirths, 2017). Figure 1. Flow-chart describing the sequential extraction procedure
Materials and Reagents
Equipment
Procedure
Notes:
Data analysis
The protein fractions obtained by using this protocol can be used in down-stream applications like Western-blot. Successful enrichment of crude synaptosomes and mitochondria can be e.g., demonstrated by Western blot analysis using marker proteins like post-synaptic density protein 95 (PSD95) or cytochrome c oxidase (CoxIV) (Saul and Wirths, 2017) (Figure 3). Figure 3. Exemplary Western blot using antibodies against PSD95 and COXIV with β-actin as loading control
Recipes
Acknowledgments
Financial support of the Alzheimer Forschung Initiative e.V. is gratefully acknowledged. This protocol was adapted from Gurd et al. (1974) with modifications.
References
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