Phospho explorer antibody microarray

Manufactured by Full Moon BioSystems

Sourced in United States

The Phospho Explorer Antibody Microarray is a lab equipment product designed to analyze the phosphorylation status of multiple proteins simultaneously. It utilizes an array of highly specific antibodies to detect and quantify the phosphorylation levels of various targets in a single experiment.

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Lab products found in correlation

Genepix pro 6, by Molecular Devices (3 mentions) Protein extraction buffer, by Full Moon BioSystems (3 mentions) Genepix 4000 scanner, by Molecular Devices (1 mentions) Axon genepix array scanner, by Molecular Devices (1 mentions) Antibody array assay kit, by Full Moon BioSystems (1 mentions)

16 protocols using phospho explorer antibody microarray

Phospho Explorer Antibody Microarray

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The Phospho Explorer antibody microarray, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA), contains 1,318 antibodies. Each of these antibodies includes two replicates that are printed on a coated glass microscope slide, along with multiple positive and negative controls. The antibody array experiment was performed according to an established protocol.

Jiang H.L., Sun H.F., Gao S.P., Li L.D., Hu X., Wu J, & Jin W. (2015). Loss of RAB1B promotes triple-negative breast cancer metastasis by activating TGF-β/SMAD signaling. Oncotarget, 6(18), 16352-16365.

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Phospho Explorer Antibody Microarray Protocol

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The phospho explorer antibody microarray, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA), contains 304 antibodies. Each of the antibodies has six replicates that are printed on coated glass microscope slides, along with multiple positive and negative controls. The antibody array experiment was performed by Huaying Biotechnology (Shanghai, China), according to their established protocol. Briefly, the cell lysates, which were obtained from γδT cells treated with IL-1β and IL-23 or madecassic acid, were biotinylated with an Antibody Array Assay Kit. The antibody microarray slides were first blocked in a blocking solution. Then the biotin-labeled cell lysate was placed on preblocked microarray slides. After washing, bound biotinylated proteins were detected using Cy3-conjugated streptavidin. The slides were scanned on a GenePix 4000 scanner, and the images were analyzed with GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA). Where indicated, protein phosphorylation data were confirmed by western blotting.

Yun X., Fang Y., Lv C., Qiao S., Tao Y., Dai Y, & Xia Y. (2020). Inhibition of the activation of γδT17 cells through PPARγ–PTEN/Akt/GSK3β/NFAT pathway contributes to the anti-colitis effect of madecassic acid. Cell Death & Disease, 11(9), 752.

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Phospho Explorer Antibody Microarray Analysis

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The Phospho Explorer antibody microarray, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA), contains 1318 antibodies [38 (link)]. Each of the antibodies has two replicates that are printed on a coated glass microscope slide, along with multiple positive and negative controls. The antibody array experiment was performed according to manufacturer's protocol [39 (link)–42 (link)]. We performed both antibody array and analysis using the customized service from ebiogen (Ebiogen Inc., Seoul, Republic of Korea).

Yim J.H., Yun J.M., Kim J.Y., Lee I.K., Nam S.Y, & Kim C.S. (2017). Phosphoprotein profiles of candidate markers for early cellular responses to low-dose γ-radiation in normal human fibroblast cells. Journal of Radiation Research, 58(3), 329-340.

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Phospho Explorer Antibody Microarray Protocol

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The Phospho Explorer Antibody Microarray was conducted by Full Moon BioSystems Inc. as published [12 , 60 (link)]. Briefly, whole-cell lysates were prepared from UTSCC14, UTSCC15, UTSCC45, SAS and FaDu cell cultures grown for 4 days in 0.5 mg/ml lrECM. Cells were harvested using modified RIPA buffer (50 mM Tris-HCl (pH7.4), 1% Nonidet-P40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, Complete protease inhibitor cocktail, 1 mM NaVO₄, 2 mM NaF). Lysates were transferred to Full Moon BioSystems Inc. on dry ice. The array consists of antibodies against 342 proteins and 606 phospho-sites. Proteins were labeled with biotin and placed on preblocked microarray slides. After washing, detection of total and phosphorylated proteins was conducted using Cy3-conjugated streptavidin. Expression of phosphorylated proteins was normalized to corresponding total protein expression. A table with results of the whole analysis is given in the supplement (Supplementary Table 5).

Klapproth E., Dickreuter E., Zakrzewski F., Seifert M., Petzold A., Dahl A., Schröck E., Klink B, & Cordes N. (2018). Whole exome sequencing identifies mTOR and KEAP1 as potential targets for radiosensitization of HNSCC cells refractory to EGFR and β1 integrin inhibition. Oncotarget, 9(26), 18099-18114.

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Phospho Protein Microarray Analysis

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A Phospho Explorer Antibody Microarray (Full Moon BioSystems, CA, USA) was used as per the manufacturer’s instructions. In brief, cell lysates were isolated using Protein Extraction Buffer (Full Moon BioSystems). After biotin labeling, the samples were applied to pre-blocked microarray slides and washed. Protein signal was detected by using Cy3-conjugated streptavidin. Signal intensities were normalized to the average intensity of the total spots on the array, and the average median signal was used for further analysis.

Mulder S.E., Dasgupta A., King R.J., Abrego J., Attri K.S., Murthy D., Shukla S.K, & Singh P.K. (2020). JNK Signaling Contributes to Skeletal Muscle Wasting and Protein Turnover in Pancreatic Cancer Cachexia. Cancer letters, 491, 70-77.

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Phospho-Specific Antibody Microarray Analysis

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The Phospho Explorer Antibody Microarray was conducted by Full Moon BioSystems Inc. (Sunnyvale, CA, USA). Whole-cell lysates from cell cultures treated with H₂O₂ (300 μM) were harvested 6 h after treatment using a Protein Extraction Buffer (Full Moon BioSystems Inc.) and transferred to Full Moon BioSystems Inc., on dry ice. The array consists of 1318 phospho-specific antibodies. In brief, proteins were labeled with biotin and placed on pre-blocked microarray slides. After washing, the detection of total and phosphorylated proteins was conducted using Cy3-conjugated streptavidin. The expression of phosphorylated proteins was normalized to the corresponding total protein expression. Fold change was calculated as follows: phosphorylation of H₂O₂-treated cells/ phosphorylation of untreated cells. This experiment was carried out once. Where indicated, protein phosphorylation data were confirmed by western blotting.

Zhang Z., Xue H., Dong Y., Zhang J., Pan Y., Shi L., Xiong P., Zhu J., Li W., Zheng W., Liu J, & Du J. (2019). GKN2 promotes oxidative stress-induced gastric cancer cell apoptosis via the Hsc70 pathway. Journal of Experimental & Clinical Cancer Research : CR, 38, 338.

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Phospho Explorer Antibody Microarray Protocol

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The Phospho Explorer Antibody Microarray was conducted by Full Moon BioSystems (Sunnyvale, CA). Whole-cell lysates from H292-sh-scramble and H292-sh-cancer-IgG treated with 6-Gy irradiation were harvested using a protein extraction buffer (Full Moon BioSystems). The protein microarray experiment was performed by Wayen Biotechnology (Shanghai, China) according to their established protocol. The fluorescence signal of each antibody was obtained from the fluorescence intensity of this antibody spot. A ratio computation was used to measure the extent of protein phosphorylation. The phosphorylation ratio was calculated as follows: phosphorylation ratio = phospho value ÷ unphospho value.

Yang X., Wang G., You J., Gu R., Xu X., Xu C., Wang H., Zhao R., Qiu X, & Zhu G. (2021). High Expression of Cancer-IgG Is Associated With Poor Prognosis and Radioresistance via PI3K/AKT/DNA-PKcs Pathway Regulation in Lung Adenocarcinoma. Frontiers in Oncology, 11, 675397.

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Phosphoproteomic Profiling of IKK/NF-κB Pathway

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The Phospho Explorer antibody microarray from Full Moon Biosystems, Inc. (Sunnyvale, CA) was used to survey the phosphorylation state of select proteins involved in the IKK/NF-κB activation pathway, according to the protocol outlined in Bernier et al. (Bernier et al., 2011 (link)). In brief, wild-type and Sirt1-KO murine embryonic fibroblasts (gift of Raul Mostoslavsky, Harvard Medical School, Boston, MA) were incubated with vehicle or 3 µM SRT1720 for 18 h, after which cell lysates were prepared and biotinylated. The antibody array experiment was performed by Full Moon Biosystems, Inc., and the data was analyzed with GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA). Each of the 1318 antibodies had two replicates printed on a coated microscope slide, along with multiple positive and negative controls.

Mitchell S.J., Martin-Montalvo A., Mercken E.M., Palacios H.H., Ward T.M., Abulwerdi G., Minor R.K., Vlasuk G.P., Ellis J.L., Sinclair D.A., Dawson J., Allison D.B., Zhang Y., Becker K.G., Bernier M, & de Cabo R. (2014). The SIRT1 activator SRT1720 extends lifespan and improves health of mice fed a standard diet. Cell reports, 6(5), 836-843.

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Phospho Explorer Antibody Microarray

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U343MG cells were cultured on type I collagen for 24 h and treated either with AIIB2/DMSO (10 μg/ml), IgG/SP600125 (EC10), AIIB2/SP600125 (EC10) or control IgG/DMSO. Cells were harvested after 1 h and whole cell lysates prepared. The Phospho Explorer Antibody Microarray was conducted by Full Moon BioSystems Inc., as previously published [12 (link)]. Additional details are described in the Supplementary Methods.

Vehlow A., Klapproth E., Storch K., Dickreuter E., Seifert M., Dietrich A., Bütof R., Temme A, & Cordes N. (2017). Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting. Oncotarget, 8(30), 49224-49237.

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Phospho-proteomic Profiling of mPDAC Cell Lines

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mPDAC cells 9091 and 8661 were plated in 10 cm dishes. The next day, they were treated for 6 hours with DMSO (vehicle) or T/N (10 nM trametinib and 2 μM nintedanib) and analyzed using the Phospho Explorer antibody microarray, which contains 1,318 antibodies (Full Moon Biosystems), according to the protocol of the manufacturer.
Pathway enrichment analysis was based on the Reactome gene-set and performed through Cytoscape (v3.8.2) with ClueGO (v2.5.8)^{66 (link)}, a Cytoscape plug-in to decipher functionally grouped pathway annotation networks. The functionally grouped networks used for visualization present terms as nodes and are linked based on their kappa score level (≥0.4). The node size represents the term-enrichment significance and functionally related groups are depicted by similar colors.

Falcomatà C., Bärthel S., Widholz S.A., Schneeweis C., Montero J.J., Toska A., Mir J., Kaltenbacher T., Heetmeyer J., Swietlik J.J., Cheng J.Y., Teodorescu B., Reichert O., Schmitt C., Grabichler K., Coluccio A., Boniolo F., Veltkamp C., Zukowska M., Vargas A.A., Paik W.H., Jesinghaus M., Steiger K., Maresch R., Öllinger R., Ammon T., Baranov O., Robles M.S., Rechenberger J., Kuster B., Meissner F., Reichert M., Flossdorf M., Rad R., Schmidt-Supprian M., Schneider G, & Saur D. (2022). Selective multi-kinase inhibition sensitizes mesenchymal pancreatic cancer to immune checkpoint blockade by remodeling the tumor microenvironment. Nature cancer, 3(3), 318-336.

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