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Assay Type
Solid Phase Sandwich ELISA
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Format
96-well strip plate
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Assay Length
4 hours 40 mins (after plate preparation)
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Sample Type & Volume Required
Cell lysates (100 µL)
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Range
62.50 - 4,000 pg/mL
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Sufficient Materials
Kits available for two, five, or fifteen 96-well plates*
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Specificity
Please see the
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure in cell lysates. An immobilized capture antibody specific for binds both phosphorylated and unphosphorylated . After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.
Product Features
- Optimized capture and detection antibody pairings with 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
- Streptavidin-HRP
Other Reagents Required
PBS: (Catalog # ), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na
2HPO
4, 1.5 mM KH
2O
4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # ), or equivalent
Lysis Buffer* IC Diluent*
Blocking Buffer* Substrate Solution: 1:1 mixture of Color Reagent A (H
2O
2) and Color Reagent B (Tetramethylbenzidine) (Catalog # )
Stop Solution: 2 N H
2SO
4 (Catalog # )
Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # ), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # ), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Data Examples
| Figure 1: The Human Total Axl DuoSet® IC ELISA is more sensitive than immunoprecipitation (IP)-Western Blot analysis. Lysates prepared from the human glioblastoma cell line A172 were diluted in series and analyzed by (A) IP-Western Blot and (B) this DuoSet® IC ELISA. IPs were performed using an anti-Axl monoclonal antibody and goat anti-mouse agarose. Immunoblots were incubated with a Biotinylated Anti-Axl Polyclonal Antibody (Catalog # ) to detect human total Axl. Bands were visualized with Streptavidin-HRP A (Catalog # ) followed by chemiluminescent detection. Human Axl can be detected in this DuoSet® IC ELISA by using approximately 10 to 20 times less lysate than is needed for a conventional IP-Western Blot. |
| Figure 2: The Human Total Axl DuoSet® IC ELISA measures the relative level of Axl. Lysates were prepared from the human glioblastoma cell line (A172), the human epidermoid carcinoma cell line (A431), and the human stomach cancer cell line (KatoIII). DuoSet® IC ELISA and IP-Western Blot (inset) analyses were performed using 50 μg and 100 μg of lysate, respectively. The IP-Western Blot was performed as described in Figure 1. The DuoSet® IC ELISA results correlate well with the amounts of human Axl detected by IP-Western Blot. |
Preparation and Storage
Background: Axl
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.
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Entrez Gene IDs:
558 (Human); 26362 (Mouse);
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Long Name:
Axl Receptor Tyrosine Kinase
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Aliases:
Ark; AXL oncogene; AXL receptor tyrosine kinase; AXL transforming sequence/gene; Axl; EC 2.7.10; EC 2.7.10.1; JTK11; tyrosine-protein kinase receptor UFO; Ufo; UFOoncogene AXL
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