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Sample GSM3906300 Query DataSets for GSM3906300
Status Public on Mar 27, 2020
Title G4_d1_IGF2_rep1
Sample type protein
 
Source name IGF2, G4 design 1
Organism synthetic construct
Characteristics molecule name: human IGF2
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 IGF2 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

Data table header descriptions
ID_REF
VALUE The median fluorescence intensity of the target sequence

Data table
ID_REF VALUE
AA1 29087
AA10 31028.5
AA100 30426
AA101 22764
AA102 39714
AA103 26623
AA104 21269
AA105 28122
AA106 27017
AA107 6156.5
AA108 21228
AA109 2552.5
AA11 28501
AA110 25235
AA111 20281
AA112 27555
AA113 19999
AA114 24156
AA115 26964
AA116 21461.5

Total number of rows: 2264

Table truncated, full table size 28 Kbytes.




Supplementary file Size Download File type/resource
GSM3906300_G4_085206_010_100PMT_1_288ng_IGF2.raw_signal.txt.gz 3.2 Mb (ftp)(http) TXT
Processed data included within Sample table

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