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A General EMSA (Gel-shift) Protocol
Published: Feb 5, 2011 DOI: 10.21769/BioProtoc.24 Views: 62861
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Abstract
An electrophoretic mobility shift assay (EMSA), also referred to as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. The control lane (the DNA/RNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. If the protein is capable of binding to the fragment, the lane with protein present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to the protein, which is 'shifted' up on the gel (since it has moved more slowly). Here, a protocol to carry out an EMSA assay is described.
Keywords: EMSA
Gel-shift
Binding
Materials and Reagents
- DTT (Promega Corporation, catalog number: V3151 )
- Poly-dIdC (Sigma-Aldrich, catalog number: P4929-10UN )
- 32P-labeled probe
Note: Oligo DNA probe can be synthesized ordered from IDT, a DNA synthesis company, then labeled by yourself. - BSA (Sigma-Aldrich, catalog number: 05470-5G )
- General chemicals (Sigma-Aldrich)
- 5x binding buffer (see Recipes)
- 10x TBE buffer (see Recipes)
Equipment
- Plates
- Spacers
- Clamps
- Saran wrap
- Whatman paper (GE Healthcare)
Procedure
Category
Molecular Biology > DNA > DNA-protein interaction
Biochemistry > Protein > Interaction
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