|
| Status |
Public on Jul 03, 2024 |
| Title |
PITG_19851_ME |
| Sample type |
protein |
| |
|
| Source name |
DBD IVT protein ME design
|
| Organism |
Phytophthora infestans |
| Characteristics |
dbd: Myb_DNA-binding
platform: ME
|
| Extracted molecule |
protein |
| Extraction protocol |
DBD sequences along with 50 amino acid residues on either side of the DBD in the native protein were cloned into pTH6838 or pTH7069, a modified T7-driven GST expression vector. For in vitro transcription_translation (IVT), we used 150 ng of plasmid DNA in a 15 μl in vitro transcription/ translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate to produce the proteins. For protein produced in E. coli, soluble proteins were purified using nickel resin and eluted in phosphate buffered saline (PBS) containing 10 to 20% glycerol. Proteins in inclusion bodies were fist solubilized in 2 M urea in PBS and refolding in 0.5 M arginine containing 10 to 20% glycerol.
|
| Label |
Alexa 647
|
| Label protocol |
We used Alexa 647-labeled anti-GST antibody to detect the GST-tagged DNA binding proteins.
|
| |
|
| Hybridization protocol |
We used either 150 ng of plasmid DNA in a 15 μl in vitro transcription/ translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate or proteins purified from E.coli. After a 2-h incubation at 37°C, 12.5 ul of the mix was added to 137.5 ul of protein-binding solution for a final mix of PBS/2% skim milk/0.2 mg per ml BSA/50 μM zinc acetate/0.1% Tween-20. This mixture was added to an array previously blocked with PBS/2% skim milk and washed once with PBS/0.1% Tween-20 and once with PBS/0.01% Triton-X 100. After a 1-h incubation at room temperature, the array was washed once with PBS/0.5% Tween-20/50 mM zinc acetate and once with PBS/0.01% Triton-X 100/50 mM zinc acetate. Alexa 647-labeled anti-GST antibody was added, diluted in PBS/2% skim milk/50 mM zinc acetate. After a 1-h incubation at room temperature, the array was washed three times with PBS/0.05% Tween-20/50 mM zinc acetate and once with PBS/50 mMzinc acetate.
|
| Scan protocol |
The array was imaged using an Agilent microarray scanner at 2 uM resolution. Images were scanned at two power settings: 100% photomultiplier tube (PMT) voltage (high), and 10% PMT (low). The two resulting grid images were then manually examined, and the scan with the fewest number of saturated spots was used. Image spot intensities were quantified using ImaGene software (BioDiscovery).
|
| Data processing |
We provide several scores for each 8-mer in each experiment. Median - median kmer intensity; Z-Score – transformed kmer median intensity; E-score – Enrichment Score. E-scores are a modified version of AUC, and describe how well each kmer ranks the intensities of the spots. Please see the supplementary files on the platform record (GPL11260) to match the raw data files with the array probes.
Median intensity for the 8mer sequence
|
| |
|
| Submission date |
Jun 20, 2024 |
| Last update date |
Jul 03, 2024 |
| Contact name |
Howard Judelson |
| E-mail(s) |
howard.judelson@ucr.edu
|
| Organization name |
University of California-Riverside
|
| Department |
Microbiology and Plant Pathology
|
| Street address |
Genomics Building
|
| City |
Riverside |
| State/province |
CA |
| ZIP/Postal code |
92521 |
| Country |
USA |
| |
|
| Platform ID |
GPL11260 |
| Series (1) |
| GSE270411 |
Binding specificities of transcription factors of the oomycete Phytophthorainfestans reflect conserved and divergent evolutionary patterns and function |
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