|
| Status |
Public on Dec 26, 2008 |
| Title |
CRZ1_4531.1_ArrayB |
| Sample type |
protein |
| |
|
| Source Name |
Baker's yeast CRZ1
|
| Organism(s) |
Saccharomyces cerevisiae |
| Characteristics |
Yeast CRZ1
|
| Extracted molecule |
protein |
| Extraction protocol |
Protein open reading frames, consisting of the pfam-defined DNA-binding domain and 15 amino acids of flanking sequence (or to the end of the full open reading frame) were cloned into pMAGIC1 by either RT-PCR from pooled yeast mRNA or by gene synthesis (DNA 2.0). We transferred the inserts into a T7-GST-tagged variant of pML280 following. We expressed proteins by either (i) purification from E. coli C41 DE3 cells (Lucigen), or (ii) in vitro translation reactions (Ambion ActivePro Kit) without purification.
|
| Label |
Cy3
|
| Label protocol |
T7-GST tagged proteins were not labelled after purification
|
| |
|
| 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) under LifterSlip cover slips (Erie Scientific) 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 100 nM (unless otherwise specified) 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 20°C. 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. Alexa488-conjugated rabbit polyclonal antibody to GST (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. Slides were spun dry by centrifugation at 40 g for 5 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 at 20°C 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 Alexa488-conjugated antibody (488 nm ex, 522 nm em). Separately, slides were scanned after primer extension to quantify the amount of incorporated Cy3-conjugated dUTP (543 nm ex, 570 nm em). Microarray TIF images were analyzed using GenePix Pro version 6.0 software (Molecular Devices)
|
| Description |
Yeast CRZ1_4531.1_ArrayB
|
| Data processing |
Every non-palindromic 8-mer occurs on at least 32 spots in each chamber of our universal PBM and we provide several scores for each 8-mer in each experiment: (1) Median Intensity, (2) Z-Score, and (3) Enrichment Score (E-Score). E-scores are a modified version of AUC, and describe how well each 8-mer ranks the intensities of the spots.
|
| |
|
| Submission date |
Aug 05, 2008 |
| Contact name |
Lourdes Pena-Castillo |
| E-mail(s) |
lourdes.pena@gmail.com
|
| Phone |
416 946-7838
|
| Fax |
416 978-8528
|
| Organization name |
University of Toronto
|
| Department |
Banting and Best Department of Medical Research
|
| Lab |
Hughes Lab
|
| Street address |
160 College St. Room 1350
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6796 |
| Series (1) |
| GSE12349 |
A library of yeast transcription factor motifs |
|
