NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM3452399 Query DataSets for GSM3452399
Status Public on Oct 31, 2018
Title tissue engineering 7d post surgery
Sample type protein
 
Source name sciatic nerve stump, tissue engineered nerve graft, 7d post surgery
Organism Rattus norvegicus
Characteristics tissue: sciatic nerve
treatment: tissue engineered nerve graft
time point: 7d post surgery
Treatment protocol Inverted autologous nerve (autologous group) or tissue engineered nerve with chitosan conduit and silk fibroin fillers (catheter group).
Growth protocol Rat sciatic nerve was exposed and lifted to transect a 0.6-cm-long nerve segment at the site just proximal to the division of tibial and common peroneal nerves. Nerve gap was bridged with inverted autologous nerve (autologous group) or tissue engineered nerve with chitosan conduit and silk fibroin fillers (catheter group). At 4, 7, and 14 days after nerve transection, rats were killed by decapitation and their sciatic nerve segments were collected for execution and validation of antibody array assay.
Extracted molecule protein
Extraction protocol Wash tissues with cold 1X PBS (4°C) with vortexing. Remove and discard PBS. Repeat 3 – 5 times. Add one tube of lysis beads to 10 – 40 mg of tissues, add 500 ul of Extraction Buffer. Mix rigorously by vortexing for 30 seconds. Incubate the mixture on ice for 30 seconds. Repeat vortexing 3 times. Centrifuge the mixture at 10,000 x g (14000 rpm) for 15 minutes at 4°C. Transfer the clear supernatant to a clean tube. Discard the beads.
Label Biotin/DMF
Label protocol Briefly centrifuge Biotin Reagent before use. Add 100 ul of DMF (N,N-Dimethylformamide) to 1 mg of Biotin Reagent to give a concentration of 10 ug/ul. Label this solution as Biotin/DMF. Aliquot 10 – 25 ul of lysate. Add Labeling Buffer to the sample to bring the volume to 67 ul. Add 3 ul of the Biotin/DMF solution to the sample. Incubate the mixture at room temperature for 2 hours with mixing. Add 30 ul of Stop Reagent. Incubate for 30 minutes at room temperature with mixing. Proceed immediately to the next step, or store the biotinylated sample at -80°C for future use.
 
Hybridization protocol In a tube, add 6 ml of Coupling Solution and one tube of biotinylated sample (25 ug). Vortex briefly to mix. Label it as “Protein Coupling Mix.” Place the slide in any clean well of the Coupling Chamber. Slowly pour all 6 ml of Protein Coupling Mix over the slide. Make sure the slide is completely submerged. Cover the Coupling Chamber. Incubate on an orbital shaker rotating at 35 rpm for 2 hours at room temperature. Transfer the slide to a 100x15 mm Petri dish containing 30 ml of 1X Wash Solution. Incubate on an orbital shaker rotating at 55 rpm for 10 minutes at room temperature. Discard the wash solution. Repeat the wash step 3 times. Rinse the slide extensively with Milli-Q Place the slide in a 50-ml conical tube. Fill the tube with 45 ml of water. Close the cap. Shake for 10 seconds. Discard the water. Repeat ten times. Shake off excessive water on the slide surface.
Add 60 ul of Cy3-Streptavidin (0.5 mg/ml) to 60 ml of Detection Buffer. Pour 30 ml of Cy3-Streptavidin Solution into a 100x15 mm Petri dish. Submerge the slide in the Cy3-Streptavidin solution. Incubate on an orbital shaker rotating at 55 rpm for 20 minutes at room temperature in the dark or covered with aluminum foil. Transfer the slide to a new 100x15 mm Petri dish containing 30 ml of 1X Wash Solution. Incubate on an orbital shaker set at 55 rpm for 10 minutes at room temperature. Discard the wash solution. Repeat the wash step twice. Rinse the slide extensively with Milli-Q grade water as follows: Place the slide in a 50-ml conical tube. Fill the tube with 45 ml of water. Close the cap. Shake for 10 seconds. Discard the water. Repeat ten times. Dry the slide with compressed nitrogen (or air) or by centrifugation. The slide is now ready for scanning.
Scan protocol The slides were scanned (GenePix 4000B, Axon Instruments, USA).
Description catheter-7d
Bridge nerve gap with chitosan+silk fibroin, collect nerve stump at 7 days post surgery.
Data processing The fluorescence signal of each antibody was obtained from the fluorescence intensity of this antibody spot after subtraction of the blank signal (spot in the absence of antibody). Two technical replicates were performed for each sample. Median signal intensity for each spot was extracted from array images and the average intensity for each antibody reaction with the protein was determined for replicate spots. Data were normalized utilizing the median intensity values for the 1318 antibodies on each array (Normalized data = Average Signal Intensity of Replicate Spots / Median Signal).
 
Submission date Oct 30, 2018
Last update date Oct 31, 2018
Contact name Sheng Yi
E-mail(s) syi@ntu.edu.cn
Organization name Nantong University
Street address 19 Qixiu Road
City Nantong
State/province Jiangsu, China
ZIP/Postal code 226001
Country China
 
Platform ID GPL18873
Series (1)
GSE121975 Protein phosphorylation profiling of rat sciatic nerve stumps

Data table header descriptions
ID_REF
VALUE Normalized signal intensity

Data table
ID_REF VALUE
1_1_1 69.92
1_1_2 0.98
1_1_3 1.00
1_1_4 1.14
1_1_5 1.17
1_1_6 1.31
1_1_7 0.94
1_1_8 1.06
1_10_1 1.39
1_10_2 1.34
1_10_3 1.04
1_10_4 0.64
1_10_5 1.12
1_10_6 0.98
1_10_7 1.11
1_10_8 1.81
1_11_1 1.44
1_11_2 1.23
1_11_3 0.74
1_11_4 1.13

Total number of rows: 1330

Table truncated, full table size 14 Kbytes.




Supplementary data files not provided
Processed data are available on Series record
Processed data included within Sample table

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