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Status |
Public on Dec 01, 2020 |
Title |
MYC4-bHLH-PBM.GenomeDesign |
Sample type |
protein |
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Source name |
Protein expressed in E.coli
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Organism |
Arabidopsis thaliana |
Characteristics |
protein expressed: Recombinant fusion protein MBP-MYC4
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Growth protocol |
E.coli (BL21 strain) was induced with 1 mM IPTG at OD600=0.6, and incubated for 4 hours at 28 ºC. After induction, cultures were centrifuged and cell pellets frozen at -80 ºC
|
Extracted molecule |
protein |
Extraction protocol |
Cell pellet from 25 ml cultures were resuspended in 1ml in 1 mL 1x binding buffer, sonicated (2x30 s) and centrifuged twice to obtain cleared extracts of soluble proteins.
|
Label |
Cy3
|
Label protocol |
DNA-protein complexes were incubated with 16 ug of rabbit polyclonal to MBP (Abcam, http://www.abcam.com/) in PBS–2% milk for 16 h at room temperature. Slides were washed 3x in PBS0.05% Tween 20, 3x in PBS-0.01% Triton X-100 (5 min each wash) and dried. Labeling of DNA-protein complexes was performed by incubating the microarrays with 0.4 ug of goat anti-rabbit IgG DyLight 549 conjugated (Pierce, http://www.piercenet.com/) in PBS–2% milk for 3 h at room temperature, followed by the same washes as before and the slides were then dried for scanning.
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Hybridization protocol |
The binding mixture containing the protein extract in binding buffer was adjusted to 175 µl and contained 2% milk and 0.89 µg of denatured salmon sperm DNA (ssDNA). This mixture was added to an array and incubated for 2.5 hours at room temperature and later on was washed 3 times with PBS-1% Tween 20 (5 min), 3x in PBS-0.01% Triton X-100 (5 min) and spun dry by centrifugation (1000 g).
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Scan protocol |
PBMs were scanned in a DNA Microarray Scanner at 5 µm resolution and quantified with Feature Extraction 9.0 software (Agilent Technologies)
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Description |
bHLH-PBM GenomeDesign
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Data processing |
Raw intensities were adjusted to account for spatial non-uniformities across the microarray using the script ‘normalize_agilent_array.pl’ included in the PBM Analysis Suite (http://thebrain.bwh.harvard.edu/PBMAnalysisSuite/indexSep2017.html)
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Submission date |
Jul 28, 2020 |
Last update date |
Dec 04, 2020 |
Contact name |
Jose M. Franco-Zorrilla |
Organization name |
Centro Nacinal de Biotecnologia-CSIC
|
Department |
Plant Molecular Genetics
|
Street address |
Darwin, 3
|
City |
Madrid |
State/province |
Madrid |
ZIP/Postal code |
28049 |
Country |
Spain |
|
|
Platform ID |
GPL28933 |
Series (2) |
GSE155320 |
DNA features beyond the transcription factor binding sites specify target recognition by plant bHLHs [GenomeData] |
GSE155321 |
DNA features beyond the transcription factor binding sites specify target recognition by plant bHLHs |
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