Affinity Ligands Applications

Affinity Ligands Applications

For DNA-related applications, affinity ligands are used in two classes of applications, indirect detection of targets and affinity chromatography. For indirect detection, the ligand-labeled DNA probe is incubated with the target, the latter being either in solution or previously immobilized onto a solid phase (like a membrane). After binding of probe to target, the probe-target complex is treated with a dye- or enzyme-labeled detection moiety (for example, for biotin, a dye-labeled streptavidin (1); for Dig or DNP, an alkaline phosphatase-labeled-anti-Dig/DNP antibody, followed by incubation with a fluorogenic/colorimetric substrate (2)). In many cases, since probe length averages about 30 bases, incorporating three affinity labels, spaced about 15 bases apart to minimize steric hindrance, enables maximum potential sensitivity via indirect detection (3). Besides their importance as nucleic acid probes, ligand-labeled oligos are also useful for purification of cognate DNA molecules or DNA binding proteins by specific hybridization-based affinity chromatography (4). For example, biotinylated oligos can be bound to a streptavidin matrix, while Dig-labeled-oligos can be bound to an anti-Dig antibody matrix. For either case, the purification can be accomplished in a column, spin or magnetic bead format.

References

(1) Pinkel, D., Straume, T., Gray, J.W. Cytogenetic analyses using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA (1986), 83: 2934-2938.
(2) Grzybowski, J., Will, D.W., Randall, R.E., Smith, C.A., Brown, T. Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups. Nucleic Acids Res. (1993), 21: 1705-1712.
(3) Kessler, C., Holtke, H.J., Seibl, R., Burg, J., Muhlegger, K. Nonradioactive labeling and detection of nucleic-acids I: a novel DNA labeling and detection system based on digoxigenin-anti-digoxigenin ELISA principle (digoxigenin system). Biol. Chem. Hoppe-Seyler (1990), 371: 917-927.
(4) Bianconcini, A., Lupo, A., Capone, S., Quadro, L., et al. Transcriptional activity of the murine retinol binding protein gene is regulated by a multiprotein complex containing HMGA1, p54nrb/NonO, protein-associated splicing factor (PSF) and steroidogenic factor 1 (SF1)/liver receptor homologue 1 (LRH-1). Int. J. Biochem. Cell Biol. (2009), 41: 2189-2203.