A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture.

AUTOR(ES)

Hagenbüchle, O

RESUMO

We describe a rapid and general method for isolating DNA-binding proteins in high yield from purified nuclei of animal cells. The method has been tested for the isolation of a series of different DNA-binding activities including those of transcription factors PTF1 and SP1. The rationale consists of first preparing purified nuclei from tissue or cells in culture by centrifugation over sucrose cushions. A synthetic, biotinylated oligonucleotide bearing the binding site for the protein of interest is then added directly to nuclei resuspended in binding buffer. At the end of the binding reaction, nuclei are removed by centrifugation; and protein-DNA complexes present in the postnuclear supernatant are attached to streptavidin-agarose. Two rounds of DNA-affinity chromatography are carried out to yield highly purified preparations of DNA-binding proteins.

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