|
| Status |
Public on Jun 26, 2023 |
| Title |
S1_SiScramble |
| Sample type |
protein |
| |
|
| Source name |
hepatocytes
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: SiScramble
array: A1
|
| Treatment protocol |
Hepatocytes were allowed to attach for 6h. Media was exchanged to eliminate non-viable/attached cells that were forward transfected with Lipofectamine RNAiMAX (ThermoFisher) and 100nM of siGENOME Non-Targeting siRNA Pool (Dharmacon, # D-001206-13-05), or siGENOME human NREP siRNA (Dharmacon, # M-019848-00-0005) in complete media. Media was then changed at 6h post-transfection with complete media.
|
| Growth protocol |
We used pure, highly viable, and plateable primary human hepatocytes from 5 pooled donors (ThermoFisher, #HMCPP5). Briefly, hepatocytes were seeded on 6-well collagen-treated plates containing William E media supplemented with primary hepatocyte supplements (ThermoFisher) and HepExtend supplement (ThermoFisher).
|
| Extracted molecule |
protein |
| Extraction protocol |
Total proteins were harvested from cell lysates using extraction buffer supplemented with proteinase and phosphatase inhibitors according to standard protocol.
|
| Label |
biotin
|
| Label protocol |
Proteins were covalently labeled with biotin. Free biotin molecules are then removed at the completion of labeling reactions by gel filtration.
|
| |
|
| Hybridization protocol |
After blocking non-specific binding sites on the array, an incubation chamber is mounted onto the microarray to permit the loading of 2 samples side by side on the same chip and prevent mixing of the samples. Following sample incubation, unbound proteins are washed away and the array is then probed with anti-biotin antibody that is labelled with a proprietary fluorescent dye combination.
|
| Scan protocol |
Each array produces a pair of 16-bit images, which are captured with a Perkin-Elmer ScanArray Reader laser array scanner (Waltham, MA).
|
| Description |
hepatocytes transduced with non-targeting siRNA
|
| Data processing |
Signal quantification is performed with ImaGene 9.0 from BioDiscovery (El Segundo, CA) with predetermined settings for spot segmentation and background correction. The background-corrected raw intensity data are logarithmically transformed with base 2.
|
| |
|
| Submission date |
Apr 11, 2023 |
| Last update date |
Jun 26, 2023 |
| Contact name |
Hui Pan |
| E-mail(s) |
Hui.Pan@joslin.harvard.edu
|
| Organization name |
Joslin Diabetes Center
|
| Department |
Bioinformatics and Biostatistics Core
|
| Street address |
1 Joslin Place
|
| City |
Boston |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL26730 |
| Series (1) |
| GSE229432 |
Antibody-microarrays in human primary hepatocytes lacking NREP |
|
