Capillary electrophoresis-based method to quantitate DNA-protein interactions.
A novel, rapid and simple capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) has been developed for the quantitative study of protein-DNA interactions. This method is particularly useful for the study of basic proteins, the most common of the DNA-interacting proteins. To avoid protein stickiness to the capillary walls we have introduced the use of neutral polyacrylamide that requires the use of reverse polarity. Under these conditions, excellent separation of DNA and protein-DNA complexes was obtained without the requirement of a gel matrix, thereby allowing the easy and reliable quantification of protein-DNA affinities. Analysis of the affinities of histones H2B and H4 for a synthetic oligo have been used to demonstrate the reproducibility and accuracy of this method. We have observed that H4 has a higher affinity for DNA than H2B, with half saturation fractions lying in the micromolar range.
Cancer Epigenetics Laboratory, Program of Molecular Pathology, Spanish National Cancer Research Center (CNIO), C/Melchor Fernández Almagro no. 3, E-28029, Madrid, Spain
Mario F
MF
Fraga
Esteban
E
Ballestar
Manel
M
Esteller
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D004268:DNA-Binding Proteins; D004247:DNA