title: Elimination of nonspecific bands in non-radioactive electrophoretic mobility shift assays using the digoxigenin system.
abstract: In the course of detecting nuclear transcription factors by electrophoretic mobility shift assay using digoxigenin (DIG)-labeled probes, we encountered a problem with a considerable nonspecific shift band in negative control lanes from which protein extracts were omitted. This nonspecific shift band can interfere with the detection of the desired target protein. Purification of the DIG-labeled probes by removing unincorporated DIG-labeled nucleotides did not resolve the problem. However, the introduction of an additional step of heating at 95 °C for 5 min and subsequent reannealing after DIG-labeled probe synthesis eliminated these nonspecific shift bands and allowed accurate analysis of the target protein.
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. Electronic address: saimi_tokunaga@hotmail.co.jp.
authors.forename: Saimi
authors.initials: S
authors.lastname: Tokunaga
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Stegeman
mesh_terms: D004247:DNA / Q000737:chemistry*; D004076:Digoxigenin / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006801:Humans; D016328:NF-kappa B / Q000737:chemistry*; D020218:Response Elements
chemical_list: D016328:NF-kappa B; D004247:DNA; D004076:Digoxigenin
keywords: Digoxigenin; Electrophoretic mobility shift assay; Non-radioactive labeling