A rapid affinity method for isolation and characterization of sequence specific DNA binding factor.
An affinity chromatography technique using avidin monomer and biotin interaction was used for isolating a sequence specific DNA binding factor. This approach was used for purifying a transcription factor that binds to 152 to 160 base pairs upstream of the transcription start site of adenovirus IVa2 promoter. A 432 bp IVa2 promoter fragment was isolated and was incorporated with biotin at the 5' end using Bio-11-dUTP and Klenow fragment of DNA pol I, it was then bound with factor in vitro in reactions containing hela cell extract in optimized conditions. After incubation, reactions were loaded onto an avidin monomer column. Adenovirus IVa2 transcription factor was purified by 0.3M, 0.6M Nacl in binding buffer, band retardation assays demonstrated a 12,000 fold purification of factor was obtained.
D000260:Adenoviruses, Human / Q000235:genetics*; D001360:Avidin; D001483:Base Sequence; D001710:Biotin; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000302:isolation & purification*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D001360:Avidin; D001710:Biotin