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 setup for these assays consists of mixing purified protein with a radiolabeled RNA substrate. The resulting complexes are then analyzed with non-denaturing (native) polyacrylamide gels poured between two glass plates followed by sample electrophoresis in a vertical apparatus³. While this approach has been used exhaustively to provide important insights in the biochemical mechanisms that underlie the binding of proteins to nucleic acids, it also has several limitations. For example, this basic strategy has relatively low throughput and it is not readily adaptable for applications that require analyzing many binding reactions in parallel. In addition, with the traditional vertical apparatus it is challenging to potentially monitor complexes at multiple times during electrophoresis^3,4.

Here we present an adaptation of the EMSA assay that uses native polyacrylamide gels cast in a flatbed apparatus, horizontal electrophoresis and fluorescently labeled RNA substrates^4,5,6,7. The incorporation of these relatively simple modifications to the basic strategy provides some powerful advantages. In particular, the horizontal flatbed format easily lends itself to analyzing dozens of samples simultaneously⁴. Also, for some RNA-protein complexes, such as those formed between the Bicaudal-C protein and its RNA substrate electrophoresis in a horizontal gel provides an increased ability to resolve distinct RNA-protein complexes and discriminate these from unbound RNA substrate.

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, and water to a final volume of 400 mL.
   2. Add 4 mL of 10% APS and 400 µL of TEMED. Mix well.
   3. Pour the mix into the horizontal casting tray and allow the gel to polymerize for 30 min. The gel will be approximately 2 cm thick.
      NOTE: Gels can cast in a fume hood to speed up polymerization. After polymerization, a thin layer of liquid will remain on top of the gel.
   4. Pour the liquid off and rinse with running buffer. Remove the comb(s) carefully and rinse the wells with running buffer using a syringe.
3. Preparation of running buffer.
   1. Prepare 2 L of 1x TBE running buffer for the gel box. Dilute 400 mL of 5XTBE with 1600 mL and mix. Place 1x TBE buffer in cold room to cool.
4. Pre-Running the gel
   1. Add 2 L of cold running buffer to the gel box and insert the gel.
   2. Pre-run the gel at 120 V for 1 h at 4 °C in a cold room. During this time, prepare the binding reaction.

2. Binding Reaction⁸

1. 2.1.Prepare the following reaction components.
   20 mM DTT
   10 mM BSA
   10 mM yeast tRNAs
   5X binding buffer (50 mM HEPES pH 8.0, 5 mM EDTA, 250 mM KCl, 0.2% Tween-20)
   100 nM fluorescein labeled RNA purchased commercially
   Concentrated Bicaudal-C protein
   50% glycerol in DEPC-treated H₂O
   DEPC-treated H₂O
2. Assemble the binding reaction components in an RNase free microcentrifuge tube.
   1. To the tube, add 5 µL of 10 mM BSA, 5 µL of 20 mM DTT, 5 µL of 10 mM yeast tRNAs, 10 µL of 5x Binding Buffer.
3. Add protein to a final concentration of 250 nM in 50 µL. Add water to a final volume of 45 µL.
4. Add 5 µL of fluorescently labeled RNA substrate. Use a commercially purchased 3' fluorescein labeled 32-nucleotide substrate.
5. Mix and incubate in the dark for 30 min at room temperature.

3. Analysis of Samples on the Native Horizontal Polyacrylamide Gel

1. To each reaction, add 15 µL of 50% glycerol and mix gently.
2. Carefully add 30 µL of each reaction to the gel. The amount of sample that can be loaded into a single well varies depending on the thickness of the gel and the size of the wells. We have loaded volumes as little as 15 µL and as much as 45 µL into a single well of gels created with a 24-well comb and poured to a thickness of 2 cm.
3. After connecting the power supply to the gel apparatus switch on the power and adjust the voltage. Run the gel in 4 °C at 120 V.
   NOTE: For the horizontal gel apparatus, this ends up being approximately 5 V/cm. For the vertical gel apparatus, this is 16 V/cm.
   1. To prevent damaging or bleaching of the fluorescently labeled RNA, conduct electrophoresis in the dark.
   2. Vary electrophoresis times depending upon the specifics of the RNA-protein complex being analyzed.
      NOTE: A major advantage of the horizontal EMSA analysis is that electrophoresis can be stopped, the gel imaged and then the gel can be placed back into the apparatus and electrophoresis can be continued for longer times.

4. Imaging the Gel

1. Perform analysis with any instrument designed for detection and imaging fluorescent substrates.
2. Place the gel on the imager.
3. Adjust imager settings. For fluorescein-labeled RNA ligands, use the following settings: FAM (wavelength 473 nm), 750 Photomultiplier voltage (PMT), 100 µm pixel size.
4. Scan the gel to capture image.

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

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