Optimization of Competitor Poly(dI-dC)•Poly(dI-dC) Levels is Advised in DNA-Protein Interaction Studies Involving Enriched Nuclear Proteins

Abstract

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.

The long zinc finger domain of PRDM9 forms a highly stable and long-lived complex with its DNA recognition sequence

“…PolydIdC is a low-complexity DNA substrate commonly used in DNA-binding studies to saturate other DNA-binding proteins in cell lysates (Larouche et al, 1996). switchSENSE measurements without additional polydIdC in the sample buffer, but diluted from the protein stock extract with <10 ng/μl for the highest PRDM9 concentration (low polydIdC condition), rendered consistently faster association rate constants than obtained when using 50 ng/μl polydIdC (high, constant polydIdC condition) in the sample buffer (725 to 1908 vs. 135 M −1  s −1 , respectively).…”

Inhibition of N-Terminal Lysines Acetylation and Transcription Factor Assembly by Epirubicin Induced Deranged Cell Homeostasis

“…Reactions were incubated for up to 9 hrs. Nuclear extracts from HEK293 cells were then added to the reactions along with the nonspecific competitor poly (dI-dC): poly(dI-dC) [43] and the binding reaction was further incubated for 20 min at RT. Samples were electrophoresed at constant voltage (200 V) under low ionic strength conditions (0.25 M Tris/acetate/EDTA buffer) on 6% polyacrylamide gels.…”

Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites

“…ParG affinity for different DNA sites was determined as the apparent equilibrium dissociation constant K app (defined as the concentration of protein required to generate 50% occupancy), which was estimated from binding curves, similar to previous analysis of the MetJ RHH protein (21). The term K app is used because protein concentration and activity (the effective concentration) are different (29) due to the use of a competitor in binding reactions (24,25). Results shown in figures are representative images of experiments performed at least in duplicate.…”