Nucleotídeos para análise da interação proteína-DNA/-RNA (EMSA)
Table 1: Nucleotide selection guide for nonradioactive EMSA probe labeling.
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 |
Produtos
Selected References
[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.