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.
J Neurosci Methods. 2018 Jan 1;293:226-233. doi: 10.1016/j.jneumeth.2017.10.006. Epub 2017 Oct 6.
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.
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.
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.
本文描述了一种从小鼠脑突触神经小体中分离多核糖体级分的详细、可靠的方案。该方法是研究神经元中局部蛋白质合成的重要工具。
我们将过滤法快速制备突触神经小体与多核糖体图谱分析相结合。我们提供了一个详细的方案，重点介绍了以下方面的困难和关键步骤：i）突触神经小体的制备；ii）多核糖体从突触神经小体中的分级分离；iii）从蔗糖梯度级分中提取蛋白质和 RNA。
与现有方法的比较 我们从突触神经小体中分离出多核糖体，并在未刺激条件下检测到 Mmp9、Camk2a 和 Stx1B mRNA 与多核糖体的结合。突触刺激导致富含重多核糖体的级分中 Mmp9 和 Camk2a mRNA 水平升高。我们将我们的方案与现有的方法进行了比较。
我们已经开发出一种可靠、有效的方法，可从突触神经小体中制备多核糖体级分，以研究多核糖体结合 mRNA 作为体外突触翻译的一个方面。
