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Sample GSM3906296 Query DataSets for GSM3906296
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

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

Data table
ID_REF VALUE
AA1 17066
AA10 18668.5
AA100 18733
AA101 14379
AA102 18250
AA103 15484
AA104 12495
AA105 15714
AA106 18774
AA107 6466
AA108 13679
AA109 4468
AA11 19873
AA110 15926
AA111 17017
AA112 19900.5
AA113 12773
AA114 16393
AA115 19274
AA116 15690.5

Total number of rows: 2264

Table truncated, full table size 28 Kbytes.




Supplementary file Size Download File type/resource
GSM3906296_G4_085206_008_30PMT_1_288ng_NCL2.raw_signal.txt.gz 3.2 Mb (ftp)(http) TXT
Processed data included within Sample table

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