Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Summary

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Abstract

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Introduction

Electrophoretic mobility shift assays (EMSAs) have proven to be an invaluable biochemical tool to analyze specific protein-nucleic acid interactions^1,2,3. These assays can provide important information regarding the binding affinity of proteins to RNA or DNA³, the component stoichiometry of nucleic acid-protein complexes¹ and provide important new insights about the binding specificity of RNA binding proteins via substrate competition experiments¹.

The traditional experimental ....

Protocol

1. Preparation of the Horizontal Native Polyacrylamide Gel⁴.

1. Preparation of the materials needed.
   1. For the horizontal gel apparatus, use a gel box (37 cm x 24 cm) with a 27 cm x 21 cm tray and a capacity for two 24-well combs. This setup provides a total of 48 samples that can be analyzed simultaneously.
   2. Prepare the following reagents: 40% Acrylamide-Bis 19:1, 5x TBE (Tris-Borate-EDTA pH 8.0), TEMED, and 10% APS (ammonium persulfate).
2. Generation of a 10% native acrylamide gel.
   1. In a 500 mL flask, add 80 mL of 5x TBE, 100 mL of 40% acrylamide, ....

Results

To demonstrate the power and versatility of the horizontal native gel electrophoresis we analyzed the binding of the Xenopus Bicaudal-C (Bicc1) protein to a fluorescently labeled RNA containing a Bicc1 binding site. Bicc1 proteins function as mRNA-specific translational repressors to control cell fate decisions during the maternal stages of animal development^9,10,11,

Discussion

Native polyacrylamide gels are an invaluable tool for investigating protein-RNA interactions and traditionally these gels are electrophoresed vertically^2,3. We have used a modification of the protocol that substitutes native polyacrylamide gels created and electrophoresed horizontally^1,4,6,7,15. These .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Horizontal Gel Box	OWL	N/A	Product no longer made. Similar gel boxes can be found at Thermo Scientific, A-series gel boxes. Catalog Number: A2-BP
24-well large large horizontal gel electrophoresis combs	OWL	N/A	Product no longer made. Similar gel boxes can be found at Thermo Scientific, A-series gel box combs. Catalog Number: A2-24C
Powerpac 300	Bio-Rad	1655050
Mini-Protean II Electrophoresis Cell	Bio-Rad	165-2940
InstaPAGE-19 40% 19:1 Acrylamide/Bis	IBI	IB70015
TEMED	IBI	IB70120
APS	IBI	IB70080
Yeast tRNAs	Ambion	AM7119
Fluorescein labeled RNA	IDT	N/A	Order can be made custom to length and desired sequence
EDTA tetrasodium salt hydrate	Sigma-Aldrich	E5391-1KG
HEPES	Sigma-Aldrich	H4034-500G
Tris Base Ultrapure	US Biological	T8600
Boric Acid	Fisher Scientific	BP168-500
Potassium Chloride	Fisher Scientific	BP366-1
Tween-20	Fisher Scientific	BP337-500
DEPC	Sigma-Aldrich	1609-47-8
Dithiothreitol (DTT)	Sigma-Aldrich	3483 12 3
Bovine Serum Albumin (BSA)	Sigma-Aldrich	9048-46-8