Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mRNAs

Publication date: Available online 6 October 2017
Source:Journal of Neuroscience Methods
Author(s): Bozena Kuzniewska, Magdalena Chojnacka, Jacek Milek, Magdalena Dziembowska
BackgroundHere we describe a detailed, reliable protocol for isolation of polysomal fractions from mouse brain synaptoneurosomes. This method is an important tool to study local protein synthesis in neurons.New MethodWe combined rapid preparation of synaptoneurosomes by filtration with polysome profiling. We provide a detailed protocol highlighting difficulties and critical steps of: i) preparation of synaptoneurosomes; ii) polyribosome fractionation from synaptoneurosomes; iii) extraction of proteins and RNA from sucrose gradient fractions.Resultsand Comparison with Existing Methods We fractionated polyribosomes from synaptoneurosomes and detected the association of Mmp9, Camk2a and Stx1B mRNA with polysomes in the unstimulated conditions. Synaptic stimulation led to increased levels of Mmp9 and Camk2a mRNA in the heavy polysomal fractions. We compared our protocol with existing methodsConclusionsWe have developed a reliable, effective method to prepare polyribosomal fractions from synaptoneurosomes to study polyribosomal binding of mRNAs as an aspect of synaptic translation in vitro.



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