protoarray human protein microarray v5.0 kinase substrate identification complete kit  (Thermo Fisher)

Journal: International Journal of Biological Sciences

Article Title: Identification of Novel Focal Adhesion Kinase Substrates: Role for FAK in NFκB Signaling

doi: 10.7150/ijbs.10273

Figure Lengend Snippet: Microarray screen for FAK substrates. a. Left: Autoradiograph image of control and FAK protein microarray slides. Right: Control microarray containing 1, 5, 10 and 25 ng/dot of GST or GST-Paxillin. b. Spot-pair signals from control and FAK slides block #40 (upper panels), with identified proteins, microarray positions and relative signal intensities (average pixels/pair) below. A-F, proteins with increased phosphorylation by FAK relative to control; G, positive control protein, PKCeta; H-J , proteins with equal or decreased phosphorylation levels in the FAK slide compared to control.

Article Snippet: The following reagents/kits were used: ProtoArray Human Protein Microarray v5.0 Kinase Substrate Identification Complete Kit (Invitrogen), full-length active Src (SignalChem), GST-FAK (Invitrogen), baculovirus-encoded Pyk2 (gift of Vita Golubovskaya, Roswell Park Cancer Institute [RPCI]), purified vitronectin (Advanced BioMatrix), purified GST-PTPN5[a.a.17-565] (ProteinTech), FAK inhibitor PF-573,228 (Sigma), TNFα (Abcam), Recombinant Light (Enzo Life Sciences).

Techniques: Microarray, Autoradiography, Blocking Assay, Positive Control

Journal: Journal of Neuroinflammation

Article Title: A new Purkinje cell antibody (anti-Ca) associated with subacute cerebellar ataxia: immunological characterization

doi: 10.1186/1742-2094-7-21

Figure Lengend Snippet: Probing of a commercial protein microarray revealed strong binding of the patient's sera to human ARHGAP26 (Panel A) . In accordance with this finding, binding of IgG from the patient's serum (Lane P), but not from three healthy controls (Lane C1-C3), to a recombinant human full length ARHGAP26 protein was found (Panel B). Western blotting confirmed the presence of ARHGAP26 in primate cerebellar extract (Panel C, lane 2), and incubation with the patient's CSF resulted in a band running at the same height as the ARHGAP26 band (Panel C, lane 1); the significance of the additional band recognized by the commercial antibody is unknown. The commercial ARHGAP26 antibody bound to the Purkinje cell layer and the molecular layer (Panel D) and showed a good overlay with the patient's IgG depicted in yellow (Panel E). Finally, preadsorption of the patient's CSF with the ARHGAP26 protein (Panel H), but not preadsorption with a control protein (Panel G), resulted in complete loss of binding to cerebellar tissue sections in an indirect immunofluorescence assay; panel F shows binding of IgG from a non-preadsorbed aliquot of the same CSF sample (exposure time was 2 sec in all cases to detect also low fluorescence signals).

Article Snippet: A commercially available human protein microarray (Protoarray v5.0; Invitrogen) spotted with >9000 human full length-proteins purified from a baculovirus-based expression system was probed with the patient's serum according to the manufacturer's instructions.

Techniques: Microarray, Binding Assay, Recombinant, Western Blot, Incubation, Immunofluorescence, Fluorescence

Journal: Molecules and Cells

Article Title: Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV

doi: 10.14348/molcells.2021.0167

Figure Lengend Snippet: (A) Identification of TEN1 as a binding partner for NS5A in protein microarray. (B) HEK293T cells were cotransfected with Myc-tagged NS5A and Flag-tagged TEN1 expression plasmid. At 48 h after transfection, total cell lysates were immunoprecipitated (IP) with an anti-Myc monoclonal antibody, and then bound protein was detected by immunoblot analysis with an anti-Flag monoclonal antibody. Arrowhead indicates IgG heavy chain. (C) Schematic illustration of both wild-type and mutants of HCV NS5A expression plasmid. WT, wild type; aa, amino acids. (D) HEK293T cells were cotransfected with Flag-tagged TEN1 and Myc-tagged NS5A expression plasmids. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag polyclonal antibody, and bound proteins were immunoblotted with an anti-Myc polyclonal antibody. Protein expressions of Myc-tagged NS5A and Flag-tagged TEN1 were verified by immunoblotting with an anti-Myc or anti-Flag monoclonal antibody using the same cell lysates.

Article Snippet: Firstly, ProtoArray ® Human Protein Microarray v5.0 (Invitrogen) was incubated with blocking buffer (50 mM HEPES [pH 7.5], 25% glycerol, 0.08% Triton X-100, 200 mM NaCl, 20 mM reduced glutathione, and 0.1 mM dithiothreitol [DTT]) for 1 h at 4°C.

Techniques: Binding Assay, Microarray, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot

Journal: Cells

Article Title: Protein Binding Partners of Dysregulated miRNAs in Parkinson’s Disease Serum

doi: 10.3390/cells10040791

Figure Lengend Snippet: Clinical characteristics of patients and controls in each cohort. N stands for number. Data are presented as mean ± standard deviation. The motor examination of Unified Parkinson’s Disease Rating Scale part III (UPDRS III) is given in points. NA means not available.

Article Snippet: miRNA/protein interactions were studied with the ProtoArray ® Human Protein Microarray v5.0 (ThermoFisher Scientific).

Techniques: Standard Deviation, Sequencing, Microarray

Journal: Cells

Article Title: Protein Binding Partners of Dysregulated miRNAs in Parkinson’s Disease Serum

doi: 10.3390/cells10040791

Figure Lengend Snippet: Investigation of miRNA protein binding partners with human protein microarrays. ( A ) Experimental workflow. ( B ) miRNA incubation signal on protein microarray. Signal is shown for hsa-mir4747-5p-atto488; panels on the left show the entire microarray spotted with ~9400 recombinant human proteins, the middle panel is an enlarged 484 protein spot sub-array, and the right panel is the enlarged spot for the MTIF3 protein (all proteins spotted in duplicate, sub-array positive controls boxed in red). ( C ) Shows miRNA/protein binding distribution in relation to background signal.

Article Snippet: miRNA/protein interactions were studied with the ProtoArray ® Human Protein Microarray v5.0 (ThermoFisher Scientific).

Techniques: Protein Binding, Incubation, Microarray, Recombinant