A panel of 53 tumorigenic growth factors and cytokines were measured in the circulation and lungs of vehicle- and microbead-treated mice at day 1 post-microbeads (n=5/group) and in the circulation of tumour-bearing mice at day 14 post-LLCs (n=4-5/group) by protein array according to manufacturers' instructions (Proteome Profiler Array, ARY015, R&D Systems, UK). Serum and lung samples were also prepared according to the manufacturers' instructions of the array.
Proteome profiler array
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Proteome Profiler Array is a multiplex antibody-based assay that enables the simultaneous detection and quantification of multiple protein targets in a single sample. The array provides a comprehensive overview of protein expression and activation levels across a wide range of biological pathways and processes.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Human cytokine antibody array, by Abcam (2 mentions)
Human xl cytokine array kit, by R&D Systems (2 mentions)
Protease inhibitor cocktail, by Promega (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (2 mentions)
Blue x ray film, by AGFA HealthCare (2 mentions)
Triton x 100, by Biosesang (1 mentions)
Ras 3000, by Fujifilm (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Fluorchem 8900, by Bio-Techne (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Anti cd4 fitc, by Beckman Coulter (1 mentions)
Azure c400, by Azure Biosystems (1 mentions)
Enhanced chemiluminescence detection kit, by GE Healthcare (1 mentions)
Imagequant las 4000 mini biomolecular imager, by GE Healthcare (1 mentions)
Human kidney biomarker array kit, by R&D Systems (1 mentions)
77 protocols using proteome profiler array
1
Profiling Tumor-Associated Factors
2
Phospho-Kinase Array Analysis
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H413 Vect Ct and hDsg3.myc cell lines seeded at equal density (80–90%) in 10 cm culture dishes were extracted and the phospho‐kinase array was carried out using 500 μg of total protein in each sample, according to the manufacturer’s instructions (ARY003C, Proteome Profiler™ Array; R&D Systems Europe Ltd).
3
Mouse Cytokine Profiling of Fibroblast-Derived EVs
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Mouse cytokine array was performed per the manufacturer’s guidelines (Proteome Profiler Array, R&D Systems). In brief, CM from mBMDMs treated with CO-Fib-sEVs and PH-Fib-sEVs were collected. CM (600 μL) from each sample with the appropriate amount of binding buffer were mixed and incubated overnight (16 hours) with a membrane containing 40 antibodies. Membranes were exposed to x-ray film and fixed and developed in respective solutions. Developed film was scanned and the estimated intensity of each dot was quantified by NIH ImageJ software.
4
Decellularized Tissue Cytokine Profiling
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Cytokines for adipogenesis and angiogenesis in the adipose and heart dECMs were investigated using a microarray kit (Proteome Profiler Array, R&D Systems, Minneapolis, MN, USA). Native and decellularized tissue samples were treated with 1% (v/v) Triton X-100 (Biosesang, Seongnam-si, Korea) in PBS with protease inhibitors and chopped for digestion. After homogenization, freezing, thawing, and centrifugation for 5 min to remove cell debris, supernatants containing cytokines were added to each array membrane on a shaker overnight at 4 °C. After treatment with the detection antibody, a Chemi Reagent Mix was added to the membranes. Finally, the membranes were exposed using an X-ray imaging machine (RAS-3000, Fuji, Aichi, Japan).
5
Profiling Kinase Phosphorylation in EMMPRIN Knockdown
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The human phospho-Kinase Array Kit (Proteome Profiler Array, ARY003, R&D Systems) was used to detect relative levels of phosphorylation of 46 kinase phosphorylation sites, according to the manufacturer's instructions, using total cell lysates of EMMPRIN or scrambled siRNA transfected HMEC cells treated or not with 50 ng/ml VEGF. Briefly, cell lysates diluted to 300 μg/mL of protein in a detergent- urea and phosphatase inhibitor-containing solubilizing buffer (R&D Systems) were incubated with the arrays overnight at 4°C. After washing unbound material, membranes were incubated with a cocktail of phosphosite–specific, biotinylated antibodies, and phosphorylated kinases were detected with streptavidin-horseradish peroxidase. Signals were revealed with a chemiluminescent substrate kit (ECL Dura Thermo Scientific, 34076). Independent experiments were performed in duplicates.
6
Cytokine Secretion Detection by ELISA
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To detect secreted cytokines, we utilized the Proteome profiler array (#ARY005B, R&D systems, Minneapolis, MN) and followed the manufacturer’s instructions to perform ELISA.
7
Profiling Secreted Cytokines from HBMMSCs
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Cell culture supernates form HBMMSCs were collected by centrifuging at 10,000 × g for 10 min. to remove cell debris. The membranes from human protein cytokine array kit (Proteome Profiler™ Array; R&D Systems) were blocked with blocking buffer at room temperature for one hour, and were followed by incubation with cell culture supernates overnight at 2–8°C on a rocking platform. The membranes were then washed with wash buffer, and incubated with the diluted Streptavidin‐HRP for 30 min. at room temperature. After washing, the membranes were assayed by chemiluminescence.
8
Inflammatory Protein Expression Analysis
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The differences in the expression of 111 inflammatory proteins were analysed in the serum and lung tissues, according to the instructions of the manufacture of the Proteome Profiler Array (R&D Systems; Minneapolis, MN, USA), a mouse antibody protein array kit. Briefly, the protein array membrane was first sealed in a blocking solution for 1 h and then incubated overnight with the serum or tissue protein at 4 °C. After repeated washing, the samples were incubated with an antibody for 1 h and then reacted with HRP-streptavidin for 30 min. Next, the protein array membrane was reacted with the chemical reagent for 1 min in darkness, and exposed to an X-ray film to obtain a protein array.
9
Phospho-Kinase Array Analysis of Kindlin-3
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The human phospho-Kinase Array Kit (Proteome Profiler Array, ARY003, R&D Systems, UK) was used to detect relative levels of phosphorylation of 46 kinase phosphorylation sites, according to the manufacturer's instructions, using total cell lysates of cells transfected with either Kindlin-3 or scrambled siRNA. Total cell lysates were diluted to 150 to 200 μg/mL protein in a detergent-, urea-, and phosphatase inhibitor-containing solubilizing buffer (R&D Systems) and incubated with the arrays overnight at 4°C. After washing away the unbound material, membranes were incubated with a cocktail of phosphosite–specific, biotinylated antibodies, to detect phosphorylated kinases with streptavidin-horseradish peroxidase and signals were revealed with a chemiluminescent substrate kit (ECL Dura Thermo Scientific, 34076). Independent experiments were performed in duplicate.
10
Profiling Immune Signaling in PBMC
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PBMC (4 × 10 [6 (link)]) were treated with C1q alone (25 μg/mL) [37 (link)], PfHz alone (10 μg/mL) and a combination of PfHz (10 μg/mL) + C1q (25 μg/mL) at 37 °C in complete RPMI 1640 medium. Cell lysates were harvested after C1q incubation (1 h) and PfHz incubation (4 h), followed by lysis in RIPA buffer containing protease and phosphatase inhibitors (Thermo Scientific) for 30 min on ice. Cell lysates were added to the human phosphor-immunoreceptor array membranes according to the manufacturer's protocol (Proteome Profiler Array; R&D Systems). Phosphorylated proteins were detected by pan-antiphosphotyrosine antibody conjugated to HRP. Array data were developed with X-ray film and quantified by FluorChem8900 (Alpha Innotech). Phosphorylation levels of individual analytes were determined by the average pixel density of duplicate spots; values were obtained after subtracting background signals and were normalised to positive controls. Culture supernatants were used for measurement of IL-1β, IL-6, TNF-α, LAIR1, and C1q protein levels through ELISA (see below).
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