|
| Status |
Public on Sep 15, 2011 |
| Title |
C41-KH-N-2_ME |
| Sample type |
protein |
| |
|
| Source name |
FOXP1-ES forkhead domain purified from C41 cells - replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
isoform: FOXP1-ES
domain: forkhead domain
isoform purified from: C41 cells
|
| Extracted molecule |
protein |
| Extraction protocol |
The forkhead domains were cloned as BamHI-BamHI fragments into pGEX-2T. C41 and XL1 cells were transformed with the plasmids and the expression of the GST fusion proteins was induced by IPTG at 37C for 4h. The proteins were purified using Glutathione Sepharose 4B resine according the manufacturer's instructions. The GST-fusion protein concentrations and purification qualitty were estimated by SDS-PAGE using serial dilutions.
|
| Label |
Cy5
|
| Label protocol |
T7-GST tagged proteins were not labelled after purification
|
| |
|
| Hybridization protocol |
12.5 ul of the in vitro translation mix was added to 137.5 ul of protein-binding solution for a final mix of PBS/2% skim milk/0.2 mg per ml BSA/50 uM 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 uM zinc acetate and once with PBS/0.01% Triton X 100/50 uM zinc acetate. Cy5-labeled anti-GST antibody was added, diluted in PBS/2% skim milk/50 uM zinc acetate. After a one-hour incubation at room temperature, the array was washed three times with PBS/0.05% Tween 20/50 uM zinc acetate and once with PBS/50 uM zinc acetate.
|
| Scan protocol |
Protein-bound microarrays were scanned using an Agilent microarray scanner at 2 uM resolution to detect Cy5-conjugated antibody.
|
| 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 sequence in each experiment: (1) Median Intensity. (2) Z-Score – transformed kmer median intensity. (3) 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 |
Jul 28, 2011 |
| Last update date |
Nov 27, 2022 |
| Contact name |
Xinchen Wang |
| E-mail(s) |
xinchenw@mit.edu
|
| Organization name |
MIT
|
| Street address |
32 Vassar St, D514
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL11260 |
| Series (2) |
| GSE30992 |
An Alternative Splicing Switch Regulates Embryonic Stem Cell Pluripotency and Reprogramming |
| GSE31007 |
An Alternative Splicing Switch Regulates Embryonic Stem Cell Pluripotency and Reprogramming [protein binding microarray] |
|
