Analysis of DNA-protein interactions using proteins synthesized in vitro from cloned genes.
To detect DNA binding activity, radiolabeled protein is incubated with specific DNA fragments, and protein-DNA complexes are separated from free protein by electrophoresis in native acrylamide gels. Unlike the more conventional mobility shift assay which utilizes (32)P-labeled DNA and unlabeled protein, the assay described here generally utilizes (35)S-labeled protein and unlabeled DNA. Major advantages of this method are that any desired mutant protein can be tested for its DNA-binding properties simply by altering the DNA template, and the subunit structure (e.g., dimer, tetramer) can be determined.
Struhl
K
K
Harvard Medical School, Boston, Massachusetts, USA.
D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014764:Viral Proteins / Q000235:genetics
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D014764:Viral Proteins; D004247:DNA; C070852:RNA polymerase SP6; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases