|
| Status |
Public on Dec 02, 2010 |
| Title |
ZFA5_HK_8mer |
| Sample type |
protein |
| |
|
| Source name |
Artificial zinc finger array 5, HK design
|
| Organism |
synthetic construct |
| Characteristics |
zn finger: ZFA5
platform_id design: HK
|
| Extracted molecule |
protein |
| Extraction protocol |
The zinc finger arrays were cloned as SacI-BamHI fragments into pTH5352, a modified T7-driven GST expression vector (see http://hugheslab.ccbr.utoronto.ca/supplementary-data/C2H2_modularity/ for a vector map). We expressed proteins using a PURExpress in vitro protein synthesis kit (New England BioLabs), supplemented with RNase inhibitor and 50 μM zinc acetate.
|
| Label |
Cy5
|
| Label protocol |
T7-GST tagged proteins were not labelled after purification
|
| |
|
| Hybridization protocol |
12.5 μl of the in vitro translation mix was added to 137.5 μl of protein-binding solution for a final mix of PBS/2% skim milk/0.2 mg per ml BSA/50 μM zinc acetate/0.1% Tween 20. This mixture was added to an array previously blocked with PBS/2% skim milk and washed once with PBS/0.1% Tween 20 and once with PBS/0.01% Triton X 100. After a one-hour incubation at room temperature, the array was washed once with PBS/0.5% Tween 20/50 μM zinc acetate and once with PBS/0.01% Triton X 100/50 μM zinc acetate. Cy5-labeled anti-GST antibody was added, diluted in PBS/2% skim milk/50 μM zinc acetate. After a one-hour incubation at room temperature, the array was washed three times with PBS/0.05% Tween 20/50 μM zinc acetate and once with PBS/50 μM zinc acetate.
|
| Scan protocol |
Protein-bound microarrays were scanned using an Agilent microarray scanner at 2 μM resolution to detect Cy5-conjugated antibody.
|
| Description |
platform design file: GPL11260
|
| Data processing |
Microarray TIF images were analyzed using ImaGene version 7.5 software (BioDiscovery)
We provide several scores for each 8-mer or 9-mer in each experiment. Z-Score – transformed kmer median intensity; E-score – Enrichment Score. E-scores are a modified version of AUC, and describe how well each kmer ranks the intensities of the spots.
Please see the supplementary files on the platform record (GPL11260) to match the raw data files with the array probes.
|
| |
|
| Submission date |
Dec 01, 2010 |
| Last update date |
Dec 01, 2010 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
harm.vanbakel@mssm.edu
|
| Organization name |
Mount Sinai School of Medicine
|
| Department |
Genetics and Genomic Sciences
|
| Lab |
Bakel Lab
|
| Street address |
One Gustave L. Levy Place, Box 1498
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL11260 |
| Series (1) |
| GSE25723 |
Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays |
|
