Optimization of competitor poly(dI-dC).poly(dI-dC) levels is advised in DNA-protein interaction studies involving enriched nuclear proteins.
Procedures used for investigating DNA-protein interactions, such as the electrophoretic mobility shift assay (EMSA) or DNasel footprinting, require that exogenous nucleic acids (or synthetic equivalents) be added to the reaction mixture to prevent or reduce the nonspecific interaction of nuclear proteins with the labeled probe of choice, especially when proteins are obtained from crude nuclear extracts. One of the most potent, and likely the most widely used, non-specific competitor is the synthetic polymer poly(dI-dC).poly(dI-dC). Its addition to the reaction mixture prior to crude nuclear proteins has unquestionably proven very efficient in reducing nonspecific interactions by facilitating detection of the complexes of interest. However, in certain instances, the use of crude extracts alone does not provide adequate answers and the need to further enrich such extracts becomes absolutely necessary. In this study, we provide evidence that amounts of poly(dI-dC).poly(dI-dC) well below those currently described in the literature substantially impair, or even totally prevent, the detection of specific DNA-protein complexes in EMSA when enriched, gel-fractionated or commercially purified nuclear proteins are used, therefore indicating the need to precisely optimize the amount of such a competitor in DNA-protein interaction studies.
Larouche
K
K
CHUL Research Center, Québec, Canada.
Bergeron
M J
MJ
Leclerc
S
S
Guérin
S L
SL
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D016329:Sp1 Transcription Factor / Q000378:metabolism
D009687:Nuclear Proteins; D011089:Polydeoxyribonucleotides; D016329:Sp1 Transcription Factor; C031156:poly d(I-C); D004247:DNA