A variety of cellular processes are triggered by sequence-specific binding of a protein to a nucleic acid (DNA or RNA) e.g. regulation of gene expression by transcription factor binding to specific DNA sequences or coordination of translation by RNA-binding proteins.
Electromobility shift assays (EMSA) are a prominent technique to detect the affinity of a protein to a known DNA or RNA sequence in vitro[1]. Briefly, a labeled RNA or DNA probe is incubated with a protein source (crude cell extract or recombinant protein) and subsequently separated via non-denaturing polyacrylamide gel electrophoresis. End labeling of DNA and RNA probes is generally preferred over random labeling methods due to minimized interference with protein binding. Protein-nucleic acid complexes exhibit a slower mobility compared to free nucleic acid probes thus complex formation can be identified by the altered migration pattern (shift).
Visualization of a protein-nucleic acid complex depends on type of label used for DNA/RNA probe labeling. Traditional labeling approaches rely on the enzymatic incorporation of radioisotope labels however, several non-radioactively labeled nucleotides have been successfully used for EMSA probe labeling (Tab. 1). Further enzymatically incorporable labeled nucleotides are available within our DNA Labeling → Probes & Epigenetics and RNA Labeling → Probes & Epigenetics section.
DIG: digoxigenin; ATTO680: fluorescent dye (exc.: 680 nm /em.: 700 nm); DY776: fluorescent dye (exc.: 771 nm /em.: 801 nm).
| Generation of... | Method | Labeling site | Enzyme | Labeled nucleotide | Incorporated nucleotides | Detection |
|---|---|---|---|---|---|---|
| ...DNA probe | 3'-End Labeling | 3'-OH | Terminal Deoxynucleotidyl Transferase (TdT) | Biotin-11-UTP[2] | 1-3 | Indirect via reporter enzyme or fluorophore conjugated streptavidin |
| DIG-11-ddUTP[3] | 1 | Indirect via reporter enzyme or fluorophore conjugated Digoxigenin antibody | ||||
| 5'-End Labeling | 5'-OH | T4 Polynucleotide Kinase (T4 PNK) | ATPγS[4] | 1 | Indirect via Iodacetamide modified biotin or fluorophores | |
| ...RNA probe | in vitro transcription | random | T7 RNA Polymerase | UTP-ATTO680[5,6] | multiple | Direct measurement of fluorescence |
| UTP-DY776[6] | multiple | Direct measurement of fluorescence | ||||
| 5'-End Labeling | 5'-OH | T4 Polynucleotide Kinase (T4 PNK) | ATPγS[4] | 1 | Indirect via Iodacetamide modified biotin or fluorophores |
[1] Hellmann et al. (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions. Nature Protocols 2:1849.
[2] Yan et al. (2006) Characterization of the human intestinal CD98 promoter and its regulation by interferon-γ. Am. J Physiol. Gastrointest. Liver Physiol. 292:G535.
[3] Kass et al. (2000) Non-radioactive electrophoretic mobility shift assay using digoxigenin-ddUTP labeled
probes. Dros. Inf. Serv. 83:185.
[4] Pagano et al. (2011) Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes. RNA 17:14.
[5] Besse et al. (2009) Drosophila PTB promotes formation of high-order RNP particles and represses oskar translation. Genes Dev. 23:195.
[6] Kohn et al. (2010) Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA. RNA 16 (7):1420.
