|
| Status |
Public on Jul 19, 2018 |
| Title |
E2f3 at 200 nM concentration |
| Sample type |
protein |
| |
|
| Source name |
Human E2f3
|
| Organism |
Homo sapiens |
| Characteristics |
transcription factor: E2f3
protein concentration: Protein concentration: 200nM
|
| Growth protocol |
The Life Technologies Gateway cloning system was used to insert full length human E2f1, E2f3, and E2f4 genes into the pET-60 destination vector with a C-terminal GST tag. Cells were grown in LB culture to an OD600 of 0.4, then protein expression was induced with 1mM IPTG at 30°C for 2hrs (E2f1), or 20°C overnight (E2f4). Pelleted cells were frozen, then thawed cells were lysed in PBS lysis buffer for two hours at 4°C while gently rocking. The lysate was centrifuged, and the protein was recovered from the soluble lysate using a GE GSTrap FF GST tag affinity column according to manufacturer’s instructions. This 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 a GE GSTrap FF GST tag affinity column according to manufacturer’s instructions.
|
| Label |
Alexa 488
|
| Label protocol |
Proteins were tagged with GST by cloning. Protein-bound arrays were incubated with Alexa-488-conjugated rabbit polyclonal antibody to GST (Life Technologies, A11001).
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| |
|
| 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 200 or 100 nM 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. Alexa-488-conjugated rabbit polyclonal antibody to His (Invitrogen) was diluted to 50 μg/ml 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. 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), bad spots were manually flagged and removed, and data from multiple Alexa 488 scans of the same slide were combined using masliner (MicroArray LINEar Regression) software.
|
| Data processing |
To correct for any possible non-uniformities in protein binding, we adjusted the Alexa 488 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. Next, the Seed-and-wobble algorithm was applied to compute enrichment scores (E-scores) and median intensities for all possible 8-mers.
Enrichment scores (E-scores), median intensities, and z-scores for 8-mers
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| |
|
| Submission date |
Jul 18, 2018 |
| Last update date |
Jul 19, 2018 |
| 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 |
GPL23935 |
| Series (2) |
| GSE97794 |
Divergence in DNA specificity among paralogous transcription factors contributes to their differential in vivo binding |
| GSE117349 |
Divergence in DNA specificity among paralogous transcription factors contributes to their differential in vivo binding [uPBM_E2f1E2f3E2f] |
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