Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors.
Protein-binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA-binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding-site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF-binding specificities at high resolution using such 'all 10-mer' universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day.
Berger
Michael F
MF
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Bulyk
Martha L
ML
D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D040081:Protein Array Analysis / Q000379:methods*; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA