从鼠脑突触神经小体中制备多核糖体级分，并分析多核糖体结合的 mRNAs。

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

机构信息

Laboratory of Molecular Basis of Synaptic Plasticity, Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland.

Laboratory of Molecular Basis of Synaptic Plasticity, Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland; Laboratory of RNA Biology and Functional Genomics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland.

出版信息

J Neurosci Methods. 2018 Jan 1;293:226-233. doi: 10.1016/j.jneumeth.2017.10.006. Epub 2017 Oct 6.

DOI:10.1016/j.jneumeth.2017.10.006

PMID:28993203

Abstract

BACKGROUND

Here 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 METHOD

We 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.

RESULTS

and 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 methods CONCLUSIONS: We 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.

摘要