Fluorometric assay of DNA binding proteins using class IIS endonuclease.
Fluorometric DNA-protein binding assay was developed with class IIS restriction enzyme and FITC-labeled DNA fixed on magnetic beads. We used this assay with AP1 and FokI as a case study to quantify DNA-protein binding. Fluorescent images of the treated beads showed that the transcriptional factor bound to its DNA site and inhibited the restricted digestion effectively. The affinity of AP1 to its site was estimated as a Kd value, which was approximately 10 nM.
Fukami
T
T
Tsukuba Research Laboratory, Hamamatsu Photonics K.K., 5-9-2 Tokodai, Tsukuba City, Ibaraki 300-2635, Japan.
Hirano
K
K
D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D066298:In Vitro Techniques; D007700:Kinetics; D008280:Magnetics; D008856:Microscopy, Fluorescence; D018808:Transcription Factor AP-1 / Q000032:analysis / Q000378:metabolism
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D018808:Transcription Factor AP-1; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; D016650:Fluorescein-5-isothiocyanate