Sequence variation within similar cis-elements promotes context-specific functions of two Drosophila GA-binding transcription factors (protein binding microarray data set)

To more specifically define the sequence content that favors direct binding of GAF, a genomic-context protein binding microarray (gcPBM) was used to detect GST-tagged GAF binding to double stranded DNA probes using a fluorescently conjugated anti-GST secondary antibody GPL22023 - PBM experiments were performed using a custom-designed oligonucleotide array in 4x180K format (Agilent Technologies, Inc., AMADID #037964), described in detail previously (Kuzu G, Kaye EG, Chery J, Siggers T et al. Expansion of GA Dinucleotide Repeats Increases the Density of CLAMP Binding Sites on the X-Chromosome to Promote Drosophila Dosage Compensation. PLoS Genet 2016 Jul;12(7):e1006120. PMID: 27414415). The array was converted to a double-stranded DNA array and used in PBM experiments essentially as described previously using PBS binding buffer with 50 μM zinc acetate (Berger et al., Nat Biotech, 2006 and Berger & Bulyk, Nat Protoc, 2009), except that here GST-GAF-DBD was applied to one fresh and one stripped array at a final concentration of 150 nM, and either 525 nM or 600 nM MBP, respectively. N-terminal GST-tagged protein samples were made for the protein GAF; samples were made by in vitro transcription translation (IVT). IVT reaction mixtures for the GAF construct were applied directly to the PBM microarray and incubated for 1hour. Microarray-bound protein was fluorescently labeled using Alexa488-conjugated antibodies targeting GST, and the microarray was scanned in using a standard microarray scanner. Median fluorescence intensity over eight replicate probes was reported for each unique DNA sequence on the microarray.