|
| Status |
Public on Dec 01, 2020 |
| Title |
bHLH17-bHLH-PBM.GenomeDesign |
| Sample type |
protein |
| |
|
| Source name |
Protein expressed in E.coli
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
protein expressed: Recombinant fusion protein MBP-b17
|
| 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.
|
| |
|
| 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).
|
| Scan protocol |
PBMs were scanned in a DNA Microarray Scanner at 5 µm resolution and quantified with Feature Extraction 9.0 software (Agilent Technologies)
|
| Description |
bHLH-PBM GenomeDesign
|
| 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)
|
| |
|
| 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 |
|
