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| Status |
Public on Feb 15, 2018 |
| Title |
Sequence variation within similar cis-elements promotes context-specific functions of two Drosophila GA-binding transcription factors (protein binding microarray data set) |
| Organism |
Drosophila melanogaster |
| Experiment type |
Genome binding/occupancy profiling by array
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| Summary |
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
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| Overall design |
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.
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| Contributor(s) |
Kaye EG, Booker M |
| Citation(s) |
29562179 |
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| Submission date |
Feb 15, 2018 |
| Last update date |
Mar 23, 2018 |
| Contact name |
Michael Tolstorukov |
| E-mail(s) |
tolstorukov@molbio.mgh.harvard.edu
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| Organization name |
Massachusetts General Hospital
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| Department |
Molecular Biology
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| Street address |
185 Cambridge Street
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platforms (1) |
| GPL22023 |
Agilent-037964 mre_4x180k_design_1_1211 Protein Binding Microarray (Condensed probe version) |
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| Samples (2) |
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| This SubSeries is part of SuperSeries: |
| GSE110654 |
Sequence variation within similar cis-elements promotes context-specific functions of two Drosophila GA-binding transcription factors |
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| Relations |
| BioProject |
PRJNA434200 |
