Approaches for the Identification and Characterization of RNA–Protein Interactions - Biophysical Approaches to Translational Control of Gene Expression - page 195

Chapter 10
Approaches for the Identification
and Characterization of RNA-Protein
Interactions

Saiprasad Palusa and Jeffrey Wilusz

10.1 Stabilizing Cellular RNA-Protein Interactions

for Improved Analysis

10.1.1 Introduction

While some RNA-protein interactions can be analyzed directly, it is advisable (par-
ticularly at the discovery phase) to increase the stability of these interaction to per-
mit their ready identification and characterization. This is generally approached by
using one of two methods: Nonreversible UV cross-linking (with or without the
inclusion of a photactivatable modified nucleoside) or reversible cross-linking using
formaldehyde. While both methods are presented below, please note that they are
independent approaches to the same goal of stabilizing RNA-protein interactions to
allow for more effective downstream characterization.

10.1.2 Specialized Materials

1. Cultured cells of interest.
2. UV light source (e.g., Stratalinker 2400; Stratagene).
3. 4-Thio uridine (Sigma-Aldrich).
4. 37% Formaldehyde (Sigma-Aldrich).
5. Phosphate Buffered Saline (PBS) (Cellgro, Mediatech, Inc.).
6. Glycine (Sigma-Aldrich).