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. 2018:1649:419-426.
doi: 10.1007/978-1-4939-7213-5_28.

Isolation and Characterization of Endogenous RNPs from Brain Tissues

Rico Schieweck  1 , Foong Yee Ang  1 , Renate Fritzsche  1 , Michael A Kiebler  2
Affiliations

Isolation and Characterization of Endogenous RNPs from Brain Tissues

Rico Schieweck et al. Methods Mol Biol. 2018.

Abstract

Identification of physiological target RNAs and protein interactors bound to RNA-binding proteins is a key prerequisite to understand the underlying mechanisms of posttranscriptional expression control and RNA granule assembly. Here, we describe a multistep biochemical approach to isolate endogenous ribonucleoprotein particles from brain tissues by exploiting differential centrifugation and gradient fractionation followed by immunoprecipitation with monospecific, affinity-purified antibodies directed against selected RNA-binding proteins. This protocol results in highly enriched endogenous ribonucleoprotein particles that then can be analyzed by mass spectrometry (for proteins composition) and microarray or RNA sequencing technologies (for target mRNAs).

Keywords: Barentsz; Differential centrifugation; Gradient fractionation; Immunoprecipitation; Neuronal RNA granules; Staufen2.

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Figures

Figure 1
Figure 1. Workflow for neuronal RNP isolation and subsequent downstream analysis
The S20 of brain homogenates is biochemically separated on an OptiPrep gradient (ranging from 15 % to 30 % of OptiPrep). Fractions enriched for Stau2 or Btz proteins are pooled and used for immunoprecipitation with highly specific and affinity purified antibodies (Scheme modified from [9]).
See this image and copyright information in PMC

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