Samples were fractionated by SDS-PAGE and blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried non-fat milk and then incubated overnight at 4°C with antibodies to JNK2 and phospho-JNK1/2 (R&D System), AKT2 and phospho-AKT2 (cat. AF 23151 and MAB 887; R&D System), NF-kB (cat. sc-109, Santa Cruz Biotechnology) and IKB-a (cat. ab 7545, Abcam Limited, Cambridge, UK). Anti-tubulin, or anti-vinculin antibodies (cat. T9026, and cat. V9131, SIGMA Chemicals Co.) were used as control for the protein loading. Antibody binding to blots was detected by chemiluminescence (Amersham Pharmacia Biotech., Cologno Monzese, Italy). For Proteome Profiler Antibody Arrays, cell lysates were prepared as described by the manufacturer (R&D System). Labscan imaging technology and software (GE Healthcare, Wauwatosa, Wisconsin) was used for Western immunoblot anlysis visualization. Band intensities were quantified by ImageJ software by pixel-integrated intensity.
Proteome profiler antibody array
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Proteome Profiler antibody arrays are a multiplex assay system designed to detect the relative levels of multiple protein targets simultaneously in a single sample. The arrays contain capture antibodies spotted in duplicate on a membrane, allowing for the parallel measurement of a variety of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mab 887, by R&D Systems (1 mentions)
Specific elisa kit, by R&D Systems (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Human angiogenesis array kit, by R&D Systems (1 mentions)
Human adipokine array kit, by R&D Systems (1 mentions)
Human soluble receptor array kit, by R&D Systems (1 mentions)
Ez capture mg, by ATTO Corporation (1 mentions)
Human xl oncology array kit, by R&D Systems (1 mentions)
Human apoptosis array kit, by R&D Systems (1 mentions)
Human phospho kinase array kit, by R&D Systems (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Softmax pro 7, by Molecular Devices (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Ka1877, by Abnova (1 mentions)
Mmyomag 74k, by Merck Group (1 mentions)
29 protocols using proteome profiler antibody array
1
Western Blot Profiling of Cell Signaling
2
Mg2+ Modulates Macrophage Cytokines
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The cytokines produced by THP1-derived macrophages after the stimulation of different concentrations of Mg2+ were determined by Proteome Profiler antibody arrays (R&D System) following the manufacturer’s instructions. The concentration of IL-8 and IL-1β was further confirmed by specific ELISA kits (R&D System).
3
Proteome Profiler Antibody Array Analysis
4
Protease and Inhibitor Expression Profiling
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Antibody-based arrays were used to assess the relative expression levels of 32 proteases and 35 protease inhibitors in conditioned medium (75 μg protein) as per the manufacturer’s instructions (Proteome Profiler antibody arrays, R and D Systems, UK).
5
Profiling Synovial Fluid Proteins in Joint Complications
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SF was used to survey factors utilizing protein array panels containing 155 well-categorized monoclonal antibodies to compare SF-specific protein-expression patterns in three groups (aseptic loosening, first-stage surgery, and second-stage surgery (two patients in the aseptic loosening group and three patients in the PJI group)). Proteome Profiler Antibody arrays (Cat#ARY005B and Cat#ARY012; R&D Systems, Minneapolis, Minnesota) containing 155 spots were used for simultaneous evaluation of the expression levels of multiple factors, including cytokines, chemokines, and soluble receptors in SF. The signal-intensity levels of spots in the protein array were quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland). Thereafter, candidate factors (i.e. IL-16, IL-18, CRELD2, E-selectin, lipocalin-2, and stromal-cell-derived factor 1 (SDF-1)) were selected for further measurement by ELISA (R&D Systems), with all ELISA measurements performed in triplicate. Thresholds for intra- and inter-assay coefficients of variation were set at < 15%, and the applicability of each candidate biomarker for PJI diagnosis was determined using receiver operating characteristic (ROC) curves.
6
Serum Proteomic Profiling of Angiogenesis, Receptors, and Adipokines
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Proteomic analyses of serum proteins were carried out using Proteome Profiler antibody arrays (R&D Systems, Minneapolis, MN), namely the Human Angiogenesis Array kit (ARY007), Human Soluble Receptor Array kit (ARY012), and Human Adipokine Array kit (ARY024). Each examination was performed according to the respective protocol. Briefly, serum samples for use with each kit were applied to nitrocellulose membranes with bound capture antibodies for target proteins and incubated for 24 hours in a refrigerated room followed by removing serum from the membrane and assaying immediately. Samples were analyzed using a LAS system (Ez‐Capture MG; ATTO Corp., Tokyo, Japan). Measured signals are presented as percentage of the negative control (0%) and positive control (100%). We selected proteins in an unbiased manner that met the following criteria: (1) significant difference (P < 0.05) in rate of change in value between the R group and NR group; (2) exhibit >1.5‐fold difference between before and 1 month after SFN treatment initiation; and (3) measured value >5%.
7
Proteome Profiling of Cell Types
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The characterisation of the two different cell types of specimen #322 was performed by using three different Proteome Profiler™ Antibody Arrays (all from R&D Systems, Minneapolis, MN, USA): Human Phospho-Kinase Array Kit, Human Apoptosis Array Kit and Human XL Oncology Array Kit. The procedure was performed according to the manufacturer’s instructions. The average expression of two independent experiments was used to determine expression relative to the positive controls present on each membrane.
8
Proteome Profiling of Tumor Lysates
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Tumour lysates were prepared using RIPA buffer (Sigma-Aldrich, UK) and serum samples were extracted from MC38 tumour bearing mice for analysis using Proteome Profiler Antibody arrays (R&D Systems, UK). Arrays were performed according to manufactures instructions and were visualized and quantified by chemiluminescence using the ChemiDoc XRS imaging system (Bio-Rad Laboratories).
9
Antibody Array Protein Quantification
10
Neutrophils Phosphoproteome Profiling
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Neutrophils (± LPS) with 0 or 0.5 mg/ml Pf (107 cells/5 ml) were incubated for 3 h in a 5% CO2 incubator set at 37 °C. Post incubation cells were harvested, lysed using a lysis buffer supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoScientific, Rockford, IL, USA), and assayed per instructions of the Proteome Profiler antibody arrays (R&D Systems, Inc., Minneapolis, MN, USA). The phosphorylated protein content (post staining) of the array was determined by the chemiluminescent reaction and quantified by densitometry by Chemidoc touch imaging system using the Image Lab software (v5.2.1; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The phosphorylated protein content was normalized to the total protein content (determined by Pierce BCA protein assay kit) per test condition with an appropriate background subtraction. Mean intensity values from one representative donor were plotted as heat map. The graph with the phosphorylated protein quantity was plotted with the mean and SEM of data from 6 donors. The repetitive presentation of a single protein in the data plots represents different phosphorylation sites of that particular protein.
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