Non-radioactive method to visualize specific DNA-protein interactions in the band shift assay.

Gel mobility shift analysis using P-labeled DNA probes is a powerful and fast method to analyse sequence-specific DNA-binding proteins, mainly transcription and enhancer factors. We routinely use this method to determine the quality of purified steroid hormone receptors, namely the rat glucocorticoid receptor and the rabbit progesterone receptor, with regard to DNA-binding. To overcome the variability and hazards of handling P labeled oligonucleotides, we have tested the sensitivity and usefulness of dlgoxygenin-dUTP for endlabeling, DNA-binding and gel retardation assays. Method: MTV-57, a synthetic oligonucleotide with 5' protruding ends, contains a HRE of the MMTV LTR. In order to obtain a high specific activity without affecting the binding site for steroid hormon receptors we labeled MTV-57 under the following conditions. Approximately 200 ng of MTV-57 DNA was incubated with 0.05 mM digoxygenin-dUTP (a gift from Boehringer Mannheim) in 10 mM Tris/HCl pH=7.5, 10 mM MgCl2, 50 mM NaCl, 1 mM DTE and 2 U K DNA polymerase at 37 °C in a total volume of 40 ul. After 10 min, 2 ul dGTP,