|
| Status |
Public on Feb 24, 2015 |
| Title |
AD at the age of 6month 001 |
| Sample type |
protein |
| |
|
| Source name |
cortical region of brain tissue
|
| Organism |
Mus musculus |
| Characteristics |
tissue: cortical region of brain tissue
age: 6 months
gender: Male
disease model: 5XFAD(AD)
|
| Extracted molecule |
protein |
| Extraction protocol |
Male animals were killed at 2 or 6 months of age(n=3). The cortical regions were used, and the tissues were solubilized with RIPA buffer which contains protease inhibitor, phosphatase inhibitor, and phenylmethane-sulfonylfluoride(PMSF).
|
| Label |
Cy3
|
| Label protocol |
Cy3 dyes were labeled to extracted proteins using Amersham CyDye mono-Reactive NHS Ester (GE Healthcare Cat. PA13101).
|
| |
|
| Hybridization protocol |
Before the hybridization, to reduce nonspecific binding, we added 200μl of a blocking buffer with 3% Block Ace (SCETI K.K., Japan, Cat. UK-B80) in PBST to each chamber of Agilent 2 chamber gasket slides (Cat. G2534-60002) and then incubated the slides for 4hrs with rotation. The slides were washed with PBST and incubated for 2 hrs with 25μg of Cy3-labeled proteins in a final volume of 200μl filled with PBST.
|
| Scan protocol |
After a series of thorough washing with PBST, PBS, and ddH2O, the slides were dried and the Cy3 fluorescence intensities were obtained by a confocal lase scanner (GenePix 4000B, Molecular Devices).
|
| Description |
6mo_AD_001
Cy3-labeled proteins from normal and AD mice model at the age of two month and 6month were hybridized to newly developed dendron-coated phosphokinase antibody array(DPA) in which four technical replicates of 22 phospho-site specific antibodies (overall 88 spots) are immobilized.
|
| Data processing |
Signal intensities obtained from the array experiment were converted into log2-intensities and normalized by scale normalization method as previously reported(Yang, Y.H. et al, Nucleic Acids Res, 2002). To identify differentially phosphorylated sites, we performed one-way ANOVA and median fold change test.
|
| |
|
| Submission date |
Feb 28, 2014 |
| Last update date |
Feb 24, 2015 |
| Contact name |
Hyobin Jeong |
| E-mail(s) |
h.jeong@imb-mainz.de
|
| Organization name |
IMB
|
| Street address |
Richard shirrmann Str 12.
|
| City |
Mainz |
| State/province |
Germany |
| ZIP/Postal code |
55112 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18350 |
| Series (1) |
| GSE55452 |
Age-dependent changes in phosphorylation of signaling molecules in brain tissues of Alzheimer's disease (AD) model mice |
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