PMC:4996396 / 14570-16348 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996396","sourcedb":"PMC","sourceid":"4996396","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996396","text":"2.3. 16k Protein Microarray\nThe in-house printed 16k protein array comprised 5449 recombinantly expressed proteins derived from 15,417 cDNA clones, printed in duplicates on the microarray resulting in approximately 32,000 spots. An illustrated microarray design of the 16k array can be found in the supplementary section (Supplementary Figure S1). The cDNA clones are included in the UniPEx expression library purchased from ImaGenes GmbH (Berlin, Germany). Sequences included in this library comprise clones derived from human fetal brain, T-cells, lung, and colon protein expression libraries. The library consists of clones with two differently modified pQE vectors. Protein expression from clones carrying the pQE80LSN vector is induced by isopropyl-β-D-thiogalactopyranosid (IPTG). Clones with the pQE30NST vector inserted exhibit auto-induced protein expression. Both types of vectors include an N-terminal 6xHis-tag [45]. Protein expression and purification was conducted as described in Stempfer et al. [22]. To estimate purity of the UniPEx proteins after purification with Ni-NTA, protein eluates were run on SDS-PAGE using NuPAGE® Novex 4%–12% Bis-Tris gels (Supplementary Figure S2).\nSU8 epoxide-coated glass slides were printed with contact printing technology using a NanoPrintTM LM210 (Dynamic Devices, Wilmington, DE, USA) with 48 SMP2 Stealth Microarray Printing Pins (TeleChem ArrayIt Microarray Division, Sunnyvale, CA, USA). E. coli lysate in the concentrations 0.3, 0.4, and 0.5 mg mL−1 was used as positive control (n = 281), and elution buffer (50 mM Na2HPO4, 500 mM imidazole, 0.3 M NaCl, 0.01% SDS and 0.005% NaN3) was spotted as negative control (n = 82) onto the slides. Printed protein slides were stored at 4°C protected from light until processing.","divisions":[{"label":"Title","span":{"begin":0,"end":27}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600218-21372816-69481428","span":{"begin":924,"end":926},"obj":"21372816"},{"id":"27600218-21078204-69481429","span":{"begin":1012,"end":1014},"obj":"21078204"}],"attributes":[{"subj":"27600218-21372816-69481428","pred":"source","obj":"2_test"},{"subj":"27600218-21078204-69481429","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93cdec","default":true}]}]}}