Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Type & Volume Required
Cell lysates (100 µL)
Sufficient Materials
Kits available for two, five, or fifteen 96-well plates*
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.
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 # ), 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 # ), or equivalent
Lysis Buffer*
IC Diluent*
Blocking Buffer*
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # )
Stop Solution: 2 N H2SO4 (Catalog # )
Microplates: 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 Phospho-MSP R/Ron DuoSet® IC ELISA is more sensitive than immunoprecipitation (IP)-Western Blot analysis. MDA-MB-453 human breast cancer cells were treated with 400 ng/mL recombinant human MSP (Catalog # ) for five minutes to induce tyrosine phosphorylation of MSP R. Serial dilutions of lysates were analyzed by (A) IP-Western Blot and (B) this DuoSet® IC ELISA. IPs were performed using an anti-MSP R monoclonal antibody and goat anti-mouse agarose. Immunoblots were incubated with a biotinylated anti-phosphotyrosine monoclonal antibody (Catalog # ) to detect phospho-MSP R. Bands were visualized with Streptavidin-HRP (Catalog # ) followed by chemiluminescent detection. Human phospho-MSP R can be detected in this DuoSet® IC ELISA by using approximately 6-12 times less lysate than is needed for a conventional IP-Western Blot. |
Figure 2: The Human Phospho-MSP R/Ron DuoSet® IC ELISA detects ligand-induced MSP R tyrosine phosphorylation. MDA-MB-453 human breast cancer cells were untreated or treated with 400 ng/mL recombinant human MSP for five minutes. ELISA and IP-Western Blot (inset) analyses were performed using 100 μg and 400 μg of lysate, respectively. IP-Western Blots for phospho-MSP R (p-MSP R) were performed as described in Figure 1. Blots were stripped and total MSP R was detected using a Biotinylated Anti-MSP R Polyclonal Antibody (Catalog # ). |
Figure 3: The specificity of the Human Phospho-MSP R/Ron DuoSet® IC ELISA is confirmed by receptor competition. MDA-MB-453 human breast cancer cells were treated with 400 ng/mL recombinant human (rh) MSP for five minutes. The indicated amounts of recombinant extracellular domains of human MSP R, human HGF R/Fc Chimera (Catalog # ), human IGF-I sR (Catalog # ), or human EGF R (Catalog # ) were added to 100 μg lysate and analyzed using this DuoSet® IC ELISA. Competition was observed only with recombinant human MSP R. |
Preparation and Storage
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: MSP R/Ron
Macrophage stimulating protein receptor (MSP R), encoded by the human RON and the mouse Stk genes, is one of a small family of receptor tyrosine kinases (RTKs) that also includes human Met (the receptor for hepatocyte growth factor) and chicken Sea. This family of receptors is synthesized as a single-chain precursor that is cleaved into a mature disulfide-linked heterodimer composed of an extracellular a chain and a membrane spanning beta chain with intrinsic tyrosine kinase activity.
Entrez Gene IDs:
4486 (Human); 19882 (Mouse);
Long Name:
Macrophage Stimulating Protein Receptor
Aliases:
CD136 antigen; CD136; CDw136c-met-related tyrosine kinase; EC 2.7.10; EC 2.7.10.1; macrophage stimulating 1 receptor (c-met-related tyrosine kinase); MSP R; MSP receptor; MSPR; MST1R; p185-Ron; Protein-tyrosine kinase 8; PTK8 protein tyrosine kinase 8; PTK8; Ron; RONmacrophage-stimulating protein receptor; soluble RON variant 1; soluble RON variant 2; soluble RON variant 3; soluble RON variant 4
