title: Immune Response Biomarker Profiling on ProtoArray™ Human Protein Microarrays V5.1 (HA20458)
status: Public on Jun 09 2020
contact.city: Houston
contact.first: Sujith
contact.middle: K
contact.last: Joseph
contact.email: sujithj@bcm.edu
contact.state: Texas
contact.address: 1102 Bates Avenue Suite 1770
contact.department: Pediatrics Hematology-Oncology
contact.country: USA
contact.institute: Baylor College of Medicine
contact.phone: 832-824-6836
organism: Homo sapiens
manufacture_protocol: (Provided by Thermofisher Scientific ProtoArray™ Human Protein Microarrays and Services)  Human clones were obtained from Thermo Fisher Scientific’s Invitrogen™ UltimateTM ORF (open reading frame) collection or from a Gateway™ collection of kinase clones developed by Protometrix. The nucleotide sequence of each clone was verified by full length sequencing. All clones were transferred into a system for expressing recombinant proteins in insect cells via baculovirus infection. Using a proprietary high-throughput insect cell expression system, thousands of recombinant human proteins were produced in parallel. Each protein is tagged with Glutathione-S-Transferase (GST), which enables high-throughput affinity purification under conditions that retain activity. After purification, a sample of every purified protein is checked to ensure that the protein is present at the predicted molecular weight. In addition, all proteins are printed onto arrays, and the concentration of each protein is determined. Proteins with the prefix PV are part of Thermo Fisher Scientific's PanVera kinase collection and may be His-tagged and/or produced in E. coli. Thermo Fisher Scientific's Invitrogen™ Ultimate™ ORF clones are not available for some of these kinases.  A contact-type printer equipped with 48 matched quill-type pins is used to deposit each of these proteins along with a set of control proteins in duplicate spots on 1” x 3” glass slides that have been coated with a thin layer of nitrocellulose. The printing of these arrays is carried out in a cold room under dust-free conditions in order to preserve the integrity of both samples and printed microarrays. Before releasing protein microarrays for use, each lot of slides is subjected to a rigorous quality control (QC) procedure, including a gross visual inspection of all the printed slides to check for scratches, fibers and smearing. Since each of the proteins is tagged with GST, a GST-directed antibody detects human proteins in a second QC assay. The procedure measures the amount of protein deposited in each spot. The arrays are designed to accommodate 23,232 spots. For the ProtoArray™ Human Protein Microarray v5.1, samples are printed in 110 μm spots arrayed in 48 subarrays (4400-μm2 each) and are equally spaced in vertical and horizontal directions with 22 columns and 22 rows per subarray (Figure 4). Spots are printed with a 200 μm spot-to-spot spacing. An extra 100 μm gap between adjacent subarrays in each row and an extra 500 μm gap between rows allows for quick identification of subarrays. A few of the human accessions are printed on two locations on the arrays. In such cases, results are averaged for duplicate spots at the same concentration and array location, but not across array locations