Labelling with digoxigenin DIG

Prepare the labelling reagent immediately prior to use by dissolving it at a corir centration of 2 mg/ml in DMSO. 

Add 24 xl of the reagent to the antibody solution slowly with stirring. 

Terminate the reaction by adding 0.1 ml of 0.1 M ethanolamine and incubate firr 15 min. 

Remove the excess biotinylating reagent by gel filtration in Sephadex G25 equilibrated with PBS containing 0.1% BSA. The IgG is in the first A280 peak. 

Store the labelled antibody at 4 C with 0.05% NaNg or in aliquots at -20 °C or lower. 

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Fig. 2. Diagrammatic representation of three-step digoxigenin detection. The target nucleic acid (1) is labeled with digoxigenin (Dig). Monoclonal anti digoxigenin (2) is linked via biotinylated (Bio) rabbit antimouse (3) to avidin alkaline phosphatase (4).

Probes can be differently labeled with hapten labels, for example carboxyfluorescein (6-FAM), digoxigenin (DIG) and biotin can be bound to LNA oligos. The choice of probe label depends on experimental design and the techniques available in the laboratory. The hapten label provides a template for crucial signal amplification since the FITC label on the oligo itself is not sufficient to allow detection in standard epifluorescence. In this study, the fluorescence signal was obtained with the TSA-FITC substrate, which allowed detection of miR-21 and miR-205. 

Fig. 1. Dual nucleic acid detection. The target DNAs (A and B) are denatured and hybridized with complementary DNA probes labeled with biotin (Bio ), or digoxigenm (Dig ). Biotin and digoxigenm residues are detected, respectively, with avidin peroxidase (red) and antibody to digoxigenin labeled with alkaline phosphatase (blue).

The labelling of antibodies with the low molecular weight molecules, biotin and digoxigenin (DIG), requires a secondary reagent for their detection and measurement In the case of DIG a labelled anti-DIG antibody is used while fiar biotin, labelled forms of the biotin binding proteins streptavidin and avidin are employed. 

Dig-UTP (BMB) is a UTP analog containing a steroid hapten isolated from Foxglove plants. In the Dig-UTP molecule, digoxigenin is coupled to the C-5 of uracil via an 11-atom spacer arm. Because of the bulky size of the Dig molecule, Dig-UTP labeling of some transcripts is more efficiently performed at a Dig-UTP/ UTP ratio of 0.15/0.85 instead of the usual 0.35/0.65. Dig-labeled probes/products can be detected or quantitated in situ or on membranes by means of an anti-Dig antibody coupled with a reporter enzyme (e.g., alkaline phosphatase) and of the enzymatic reaction with chromogenic or (chemi)luminescent substrates. 

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