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Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay)
Published: Vol 3, Iss 22, Nov 20, 2013 DOI: 10.21769/BioProtoc.967 Views: 30180
Edited by: Tie Liu
Original research article
The authors used this protocol in:
Apr 2013
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Abstract
RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements in nascent or mature transcripts. There are several hundreds of RBPs in plants, but the targets of most of them are unknown. A variety of experimental methods have been developed to identify targets of an RBP. These include RNA immunoprecipitation (RIP), UV cross-linking and immunoprecipitation (CLIP) and many variations of CLIP (e.g. PAR-CLIP, iCLIP). These approaches depend on immunoprecipitation of RNAs bound to a specific RBP using an antibody to that RBP. Electrophoretic mobility shift assay (EMSA), also called gel shift assay, has been used to analyze protein-nucleic acid interactions. It is a simple and powerful method to analyze protein-RNA/DNA interactions. In RNA EMSA, RNA-protein complexes are visualized by comparing the migration of RNA in the presence of a protein. Generally, in RNA EMSA a specific RNA sequence is used to analyze its interaction with a protein. In vitro transcribed 32P labeled or chemically synthesized RNA with a fluorescent tag is incubated with or without the protein of interest and the reaction mixture is then run on native polyacrylamide gel electrophoresis. RNA-Protein complexes migrate slowly as compared to free RNA, which can be visualized using an imaging system. In addition to test binding of an RBP to RNA, EMSA is also used to map the region in RNA and/or protein that is involved in interaction. Furthermore, the binding affinity can also be quantified using EMSA.
Materials and Reagents
Note: All work should be done in an RNase free environment, using only sterile, RNase free solutions and materials.
- E. coli BL21 (DE3)-pLysS host cells harboring SR45 cDNA
- In vitro transcribed RNA
- Purified recombinant protein
- Cap analogue (New England Biolabs, catalog number: S1404S )
- SP6 polymerase (Fermentas, catalog number: EP0131 20 U/μl)
- rNTPs
ATP (Fermentas, catalog number: R0441 )
CTP (Fermentas, catalog number: R0451 )
GTP (Fermentas, catalog number: R0461 )
UTP (Fermentas, catalog number: R0471 )
- RNase Inhibitor (Life Technologies, Invitrogen™, catalog number: 10777019 )
- 32P-UTP- Uridine 5’ triphosphate (PerkinElmer, catalog number: BLU007C001 )
- HEPES (Thermo Fischer Scientific, catalog number: AC172571000 )
- KCl (Sigma-Aldrich, catalog number: P9541 )
- MgCl2 (Mallinckrodt, catalog number: 5958-04 )
- Glycerol (Thermo Fischer Scientific, catalog number: G331 )
- Triton X-100 (Thermo Fischer Scientific, catalog number: BP151 )
- Sodium dodecyl sulfate (SDS) (Mallinckrodt Baker, catalog number: 4095-02 )
- NaCl (Thermo Fischer Scientific, catalog number: 7647-14-5 )
- Na2HPO4 (Mallinckrodt, catalog number: 7917-04 )
- KH2PO4 (Thermo Fischer Scientific, catalog number: 7778-77-0 )
- Spermidine (Sigma-Aldrich, catalog number: 52626 )
- Heparin sulfate (Sigma-Aldrich, catalog number: H4784 )
- Tris base (AMRESCO, catalog number: 77-86-1 )
- Acrylamide/bis-acrylamide (Bio-Rad Laboratories, catalog number: 16105B )
- Phenol: chloroform (AMRESCO, catalog number: 0883 )
- Ammonium persulfate (APS) (Gibco BRL®, catalog number: 5523UA )
- TEMED (VWR International, catalog number: 97064-684 )
- Bromophenol blue (Sigma-Aldrich, catalog number: 161-0404 )
- Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
- pGEM vector (Promega, catalog number: P2161 )
- Isopropyl-β-D thiogalactopyranoside (IPTG) (Gold Bio, catalog number: I2481C5 )
- Ampicillin (VWR International, catalog number: IB02040 )
- S-Protein agarose beads (Merck KGaA, Novagen®, catalog number: 80031-014 )
- Lysozyme (Sigma-Aldrich, catalog number: L6876 )
- pET32c vector (Merck KGaA, Novagen®, catalog number: 69017-3 )
- Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
- Boric acid (Mallinckrodt, catalog number: 2549-04 )
- Ethylenediaminetetraacetic acid (EDTA) (Thermo Fischer Scientific, catalog number: S311-500 )
- Ammonium acetate (Thermo Fischer Scientific, catalog number: A637-500 )
- Bovine serum albumin (BSA)
- LB medium
- 5x Gel shift buffer (see Recipes)
- Lysis buffer (see Recipes)
- 10x TBE (see Recipes)
- Gel Mix (see Recipes)
- 5% Denature Gel (see Recipes)
- 5% Non-Denaturing Gel (see Recipes)
- TNS Solution (see Recipes)
- RNA loading dye for Urea gel (see Recipes)
- 6x loading buffer for Non-Urea gel (see Recipes)
- Binding/wash buffers (see Recipes)
- Phosphate buffer (see Recipes)
- Citrate buffer (see Recipes)
Equipment
- 1.5 ml microcentrifuge tube (BioExpress, catalog number: C-3262 )
- Saran wrap
- Whatman filter paper
- Water bath (VWR International, model: 1225 PC )
- Electrophoresis equipment with power supply (Thermo Fisher Scientific, model: B2 easy cost )
- Gel drying apparatus (Savant System LLC, model: SGD 2000 )
- Phosphorimager (Molecular Dynamics, model: Storm 840 )
- Phosphorimager screen (Molecular Dynamics, model: 1000004864 )
- Sonicator (SP Scientific, model: 274480 )
- 0.45 micron syringe filter (Life Science Products, catalog number: 25CSO80AS )
- Centrifuge (Eppendorf, model: 022620401 )
- Vertical gel apparatus (Gibco BRL®, model: V15.17 )
- Liquid scintillation counter
- Orbital shaker
Procedure
Category
Plant Science > Plant biochemistry > Protein
Plant Science > Plant molecular biology > RNA
Molecular Biology > RNA > RNA-protein interaction
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