A short protocol for micro-purification of nuclear proteins from whole animal tissue.
Although a number of small-scale procedures have been described for the preparation of crude nuclear extracts from established cell lines, none were provided for the preparation of similar extracts from small amounts of animal tissue. In addition, no small-scale procedures contain enrichment steps that render the detection of low-abundant DNA-binding proteins easier. Here we describe a simple, efficient procedure for the rapid preparation of high-quality nuclear extracts from either whole animal tissue or established cell lines. It is based on a rapid isolation of the nuclei followed by a KCl extraction and a further micro-enrichment of the DNA binding proteins on heparin Sepharose CL-6B. Extracts prepared in such a way are suitable for the analysis of specific DNA/protein interactions by the use of gel shift assays or by DNaseI and dimethylsulfate footprinting techniques. Most importantly, the entire process can be fulfilled at minimal cost within a day on as little as one gram of fresh tissue, which renders this procedure extremely attractive for the analysis of DNA binding proteins involved in the control of gene expression.
Roy
R J
RJ
Université Laval.
Gosselin
P
P
Guérin
S L
SL
D000818:Animals; D001483:Base Sequence; D004247:DNA; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D051381:Rats; D013463:Sulfuric Acid Esters; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D013463:Sulfuric Acid Esters; D006493:Heparin; D004247:DNA; D003850:Deoxyribonuclease I; C007482:dimethyl sulfate