|
| Status |
Public on Mar 27, 2020 |
| Title |
G4_d1_NCL2_rep1 |
| Sample type |
protein |
| |
|
| Source name |
Nucleolin (residues 272-710), G4 design 1
|
| Organism |
synthetic construct |
| Characteristics |
molecule name: human NCL(residues 272-710)
dna type: ssDNA
array treatment: 100mM KCl
|
| Treatment protocol |
Microarrays were preincubated with a 100mM potassium chloride solution for 1 hour at room temperature to induce G4 formation. Protein binding microarray experiments were then performed as previously described (Badis et al., Science 2009). For experiments examining dsDNA, single-stranded DNA probes were made double-stranded using a primer complementary to a 24-mer constant sequence on the array probes following the method described previously (Badis et al., Science 2009). Double-stranding efficiency was monitored using 4% Cy3-dCTP.
|
| Extracted molecule |
protein |
| Extraction protocol |
We synthesized Cy5-labeled pyridostatin (Cy5-PDS). We obtained BG4 conjugated with FluoProbes®647H (Cy5-BG4) from Absolute Antibody (product number Ab00174-1.1). N-terminal Glutathione S-transferase (GST) tagged human nucleolin, IGF2, CNBP, BLM, and FANCJ plasmids were synthesized by GenScript.
|
| Label |
Cy5
|
| Label protocol |
All chimeric proteins were expressed via in vitro translation (IVT) reactions using the PURExpress In Vitro Protein Synthesis Kit (NEB). For all IVT reactions, 288ng of plasmid was added to 80uL of IVT mixture and reactions were carried out at 37°C for 2 hours. Expression of all protein constructs was confirmed via Western Blot.
|
| |
|
| Hybridization protocol |
Microarrays were blocked with 4% non-fat dry-milk in a potassium phosphate buffer before incubation with proteins or small molecules. The binding reactions were carried out in a hydration chamber for 1 hour followed by one wash with 0.5% Tween-20 in 1xPBS. Protein-bound arrays were incubated with Alexa Fluor 647 conjugated Anti-GST antibody for 1 hour, followed by three washes with 0.05 % Tween-20. Finally array slides were washed and dried in 1x PBS and scanned using an Agilent Sure Scan II scanner.
|
| Scan protocol |
Protein or molecule-bound microarrays were scanned with the G5761A SureScan Dx Microarray Scanner System (Agilent) to detect Cy5 signal at two laser settings (30 and 100PMT) to ensure signal intensities were below saturation. Spot intensities from microarray images were extracted using the Agilent Feature Extraction Software and are reported as raw fluorescence units. Microarrays with the fewest number of saturated spots were used for further analysis.
|
| Description |
human NCL(residues 272-710) G4 array experiment, ssDNA + 100mM KCl
|
| Data processing |
An intensity value was calculated for each target sequence by computing the median signal intensity across probes containing an identical probe identifier (ProbeID)
|
| |
|
| Submission date |
Jun 26, 2019 |
| Last update date |
Mar 27, 2020 |
| Contact name |
Desiree Tillo |
| E-mail(s) |
desiree.tillo@nih.gov
|
| Phone |
+1-240-760-7289
|
| Organization name |
NIH/NCI
|
| Street address |
41 Center Dr, Room D310
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL26848 |
| Series (2) |
| GSE133365 |
Custom DNA microarrays reveal diverse binding preferences of proteins and small molecules to thousands of G-quadruplexes [design 1] |
| GSE133368 |
Custom DNA microarrays reveal diverse binding preferences of proteins and small molecules to thousands of G-quadruplexes |
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