Human Phospho-Mer DuoSet IC ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Product Features
- Optimized capture and detection antibody pairings and recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15-(96-well) plate pack sizes
- Economical alternative to Western blot
Kit Content
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Lysis Buffer*
IC Diluent*
Blocking Buffer*
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Scientific Data
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The Human Phospho-Mer DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western analysis NS0 cells transfected with human Mer (NS0-hMer) were treated with conditioned media from the human lung cell line, WI-38, for 15 minutes to induce tyrosine phosphorylation of Mer. Lysates were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet IC ELISA. IPs were done using an anti-Mer monoclonal antibody and goat anti-mouse agarose. Immunoblots were incubated with an HRP-conjugated anti-phosphotyrosine monoclonal antibody (R&D Systems, Catalog # HAM1676) to detect phospho-Mer. Bands were visualized by chemiluminescent detection using WesternGloTM Chemiluminescent Detection Substrate (R&D Systems, Catalog # AR004). Human Phospho-Mer can be detected in this DuoSet IC ELISA by using approximately 5 to 10 times less lysate than is needed for a conventional IP-Western blot.
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The Human Phospho-MerDuoSet IC ELISA detects ligand-induced Mer tyrosine phosphorylation NS0-hMer cells were untreated or treated with conditioned media from the human lung cancer cell line, WI-38, for 15 minutes. ELISA and IP-Western blot (inset) analyses were done using 100 μg and 400 μg of lysate, respectively. IP-Western blots for phospho-Mer (p-Mer) were done as described in Figure 1. Blots were stripped and total Mer was detected using a biotinylated anti-Mer polyclonal antibody (R&D Systems, Catalog #BAF891).
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The specificity of the Human Phospho-MerDuoSet IC ELISA is confirmed by receptor competition NS0-hMer cells were treated with conditioned media from the human lung cell line, WI-38, for 15 minutes minutes. The indicated amounts of recombinant extracellular domains of human Mer/Fc (R&D Systems, Catalog #891-MR), human Axl/Fc (R&D Systems, Catalog #154-AL), human Dtk/Fc (R&D Systems, Catalog #859-DK) or human TrkA/Fc (R&D Systems, Catalog #175-TK) were added to 100 μg lysate and analyzed using this DuoSet IC ELISA. Competition was observed only with recombinant human Mer.
Product Datasheets
Preparation and Storage
Background: Mer
Axl (Ufo, Ark), Dtk (Sky, Tyro3, Rse, Brt) and Mer (human and mouse orthologs of chicken c-Eyk) constitute the TAM receptor tyrosine kinase subfamily. This RTK subfamily is characterized by an extracellular domain that consists of two Ig-like motifs and two fibronectin type III motifs. These receptors bind the vitamin K-dependent protein Growth Arrest Specific Gene 6 (Gas6). Receptor activation leads to cell proliferation, migration, or the prevention of apoptosis. Cellular signaling through this family of RTKs is involved in hematopoiesis, embryonic development, tumorigenesis, and spermatogenesis.
FAQs
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Which phosphorylated sites are recognized by this assay?
This assay utilizes an anti-phosphorylated tyrosine monoclonal detection antibody, and it recognizes all phosphorylated tyrosine residues.
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Induction of phospho-MerTK in NCI-H1299 cells at 20 minute with a selective, potent anti-MerTK inhibitor.
