|
| Status |
Public on Jul 30, 2023 |
| Title |
Ets1 binding levels |
| Sample type |
protein |
| |
|
| Source name |
Human Ets1
|
| Organism |
synthetic construct |
| Characteristics |
transcription factor: Ets1
ets1 concentration: 1nM
runx1 concentration: 0nM
|
| Growth protocol |
Gateway-compatible clones containing the full-length genes for Ets1 and Runx1 were respectively transferred into pDEST15 vector using the LR Clonase reaction (Life Technologies). pDEST15 vector enables the production of N-terminally GST-tagged proteins. Cells were grown in LB broth to an OD600 of 0.4 to 0.6 and then expression was induced with IPTG at 30° to 37° C. Pelleted cells were frozen, stored at -20 C, and thawed cells were lysed with lysozyme.
|
| Extracted molecule |
protein |
| Extraction protocol |
GST-tagged protein was purified from the soluble portion of the lysate using GST resin (GE Healthcare) according to manufacturer’s instructions.
|
| Label |
Alexa 488
|
| Label protocol |
Proteins were tagged with N-terminal GST by cloning. Protein-bound arrays were first incubated with primary antibody (CST 14069 for Ets1, Abcam 23980 for Runx1), and then Alexa-488-conjugated donkey-anti-rabbit secondary antibody (Invitrogen).
|
| |
|
| Hybridization protocol |
Double-stranded microarrays were first pre-moistened in PBS / 0.01% Triton X-100 for 5 min and blocked with PBS / 2% (wt/vol) nonfat dried milk (Sigma) for 1 h. Microarrays were then washed once with PBS / 0.1% (vol/vol) Tween-20 for 5 min and once with PBS / 0.01% Triton X-100 for 2 min. Proteins were diluted to desired concentrations in a 175-μl protein binding reaction containing PBS / 2% (wt/vol) milk / 51.3 ng/μl salmon testes DNA (Sigma) / 0.2 μg/μl bovine serum albumin (New England Biolabs). Preincubated protein binding mixtures were applied to individual chambers of a four-chamber gasket cover slip in a steel hybridization chamber (Agilent), and the assembled microarrays were incubated for 1 h at room temperature. Microarrays were again washed once with PBS / 0.5% (vol/vol) Tween-20 for 3 min, and then once with PBS / 0.01% Triton X-100 for 2 min. Primary antibody to Ets1 (CST) or Runx1 (Abcam) was diluted with a dilution rate of 1:50 in PBS / 2% milk and applied to a single-chamber gasket cover slip (Agilent), and the assembled microarrays were again incubated for 1 h at 20°C. Alexa488-conjugated secondary antibody targeting the primary antibody was then applied with a dilution rate of 1:50. Finally, microarrays were washed twice with PBS / 0.05% (vol/vol) Tween-20 for 3 min each, and once in PBS for 2 min. After each hour-long incubation step, microarrays and cover slips were disassembled in a staining dish filled with 500 ml of the first wash solution. All washes were performed in Coplin jars on an orbital shaker at 125 r.p.m. Immediately following each series of washes, microarrays were rinsed in PBS (slowly removed over approximately 10 seconds) to ensure removal of detergent and uniform drying.
|
| Scan protocol |
Protein-bound microarrays were scanned to detect Alexa-488-conjugated antibody (488 nm ex, 522 nm em) using at least three different laser power settings to best capture a broad range of signal intensities and ensure signal intensities below saturation for all spots. Microarray TIF images were analyzed using GenePix Pro version 6.0 software (Molecular Devices).
|
| Data processing |
To correct for any possible non-uniformities in protein binding, we adjusted the fluorescence signals according to their positions on the microarray. We calculated the median normalized intensity of the 15 x 15 block centered on each spot and divided the spot's signal by the ratio of the median within the block to the median over the entire chamber. For each unique sequence represented on the array, we then calculated the median over replicate spots and then normalized the signal for direct comparison between experiments.
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| |
|
| Submission date |
Apr 08, 2021 |
| Last update date |
Jul 30, 2023 |
| Contact name |
Raluca Gordan |
| E-mail(s) |
raluca.gordan@duke.edu
|
| Organization name |
Duke University
|
| Department |
Center for Genomic and Computational Biology
|
| Street address |
101 Science Dr, CIEMAS 2179
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
| |
|
| Platform ID |
GPL29992 |
| Series (2) |
| GSE171734 |
High-throughput data and modeling reveal insights into the mechanisms of cooperative DNA-binding by transcription factor proteins (ETS1 and RUNX1) |
| GSE171735 |
High-throughput data and modeling reveal insights into the mechanisms of cooperative DNA-binding by transcription factor proteins |
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