NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM2752104 Query DataSets for GSM2752104
Status Public on Aug 23, 2018
Title T7 primase (residue: 1-271) at 5 uM concentration
Sample type protein
 
Source name T7 primase
Organism Escherichia phage T7
Characteristics protein: T7 primase
protein concentration: 5uM
Growth protocol We have used T7 primase containing His tag at the N terminus. The T7 primase domain (residue: 1-271) was constructed, over-produced and purified as previously described (Zhu B, Lee S-J, Richardson CC JBC 2009 284(35)23842). Briefly, the coding region of T7 gene 4 primase domain was inserted into pET28b (EMD Biosciences), and the coding region in this plasmid was confirmed by DNA sequencing.
Extracted molecule protein
Extraction protocol Extraction protocol is based on a modified protocol described before (Ilic S et al., 2016 Sci. Reports), except that the purification step was based upon Ni-NTA purification and a subsequent step of gel filtration. For expression of His-tagged protein, a starter culture was used to inoculate 1 L of LB medium containing 50 µg/mL kanamycin. When the culture optical density (600 nm) reached approximately 0.7, it was induced for protein overexpression with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 h at 37°C. The cells were harvested by centrifugation (4,700 x g for 20 minutes). The cell pellet was resuspended in buffer A (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1mM DTT) containing 0.25 mg/ml Lysozyme and 1 mM PMSF and incubated on ice for 1 h. After four freeze-thaw cycles, streptomycin sulfate was added until it reached a concentration of 1%. After centrifugation at 15,000 × g for 30 minutes, supernatant was diluted with buffer A and loaded onto a prepacked Ni-NTA affinity column (GE Healthcare) in the presence of 10 mM imidazole. T7 primase bound to the Ni-NTA column was eluted using buffer A containing a gradient of imidazole from 10 to 500 mM. Fractions containing primase were combined and ammonium sulfate was added (0.4 g/ml). The precipitate was dissolved in 5 mL buffer A and was loaded onto a superdex S-200HR column and eluted using Buffer B (50 mM Tris-HCl pH 7.5, 0.1mM DTT, 1mM EDTA, 50mM NaCl). The fractions containing purified T7 DNA primase were combined and dialyzed against buffer C (50mM Tris-HCl pH 7.5, 1mM DTT, 1mM EDTA, 50% glycerol) and stored at -20oC. The purified His tagged protein was ~95% pure as determined by SDS-PAGE analysis and has comparable activity as the wild type gene 4 protein. In addition, it has been shown previously that the tag at the N-terminus supports the growth of T7∆4 phage (Zhu B, Lee S-J, Richardson CC JBC 2009 284(35)23842), thus the tag does not affect our analysis.
Label Alexa 488
Label protocol Proteins were tagged with N-terminal His by cloning. Protein-bound arrays were incubated with Alexa-488-conjugated rabbit polyclonal antibody to His (Invitrogen).
 
Hybridization protocol no hybridiztion
Scan protocol Protein-bound microarrays were scanned to detect Alexa-488-conjugated antibody (488 nm ex, 522 nm em) using at least three different laser power settings to best capture a broad range of signal intensities and ensure signal intensities below saturation for all spots. Microarray TIF images were analyzed using GenePix Pro version 6.0 software (Molecular Devices), bad spots were manually flagged and removed, and data from multiple Alexa 488 scans of the same slide were combined using masliner (MicroArray LINEar Regression) software.
Data processing None
 
Submission date Aug 23, 2017
Last update date Aug 23, 2018
Contact name Raluca Gordan
E-mail(s) raluca.gordan@duke.edu
Organization name Duke University
Department Center for Genomic and Computational Biology
Street address 101 Science Dr, CIEMAS 2179
City Durham
State/province NC
ZIP/Postal code 27708
Country USA
 
Platform ID GPL23947
Series (1)
GSE102981 Sequence context controls the DNA binding and processivity of the T7 DNA primase

Data table header descriptions
ID_REF
VALUE median signal intensity over replicate spots (6 replicates per DNA sequence)

Data table
ID_REF VALUE
T7_00001_G01 5280.5
T7_00002_G01 1939.5
T7_00003_G01 2143
T7_00004_G01 2105
T7_00005_G01 5432.5
T7_00006_G01 2296.5
T7_00007_G01 1897
T7_00008_G01 3764
T7_00009_G01 4415
T7_00010_G01 2425.5
T7_00011_G01 4339.5
T7_00012_G01 4660
T7_00013_G01 2013.5
T7_00014_G01 2071
T7_00015_G01 5400
T7_00016_G01 2230.5
T7_00017_G01 2496.5
T7_00018_G01 5118
T7_00019_G01 2174.5
T7_00020_G01 2203

Total number of rows: 29317

Table truncated, full table size 549 Kbytes.




Supplementary file Size Download File type/resource
GSM2752104_T7primase_RawData.txt.gz 11.2 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap