Using proteases to avoid false identification of DNA-protein complexes in gel shift assays
Lee, T.C.; Schwartz, R.J.
Biotechniques 12(4): 486-490
1992
ISSN/ISBN: 0736-6205 PMID: 1503744 Document Number: 390878
Gel mobility shift assays using crude nuclear extracts may result in the formation of multiple DNA-protein complexes reflected by their discrete gel mobilities. Identification of the multiple complexes can sometimes be complicated by the presence of protease activities in the extract as demonstrated here. We describe a simple protease-mediated partial digestion method that can be coupled with the gel shift assay to overcome the problem. The combined approach enables us to identify gel complexes that arise from protein degradation and therefore is suitable for analyzing those DNA-binding proteins exhibiting prominent protease sensitivity. The method should prove particularly informative in the search for tissue-specific complexes when crude extracts from different sources are compared by the gel shift assay.