SH2 Domain-Based RTK Profiling Kit

Receptor tyrosine kinases (RTKs) play important roles in converting extracellular to intracellular signals. Total 58 RTKs are identified in the human genome. They are activated by the binding of specific growth factors, cytokines, or hormones, which results in autophosphorylation. Tyrosine autophosphorylation generates recruitment sites for downstream signaling proteins containing Src homology-2 (SH2). Binding of different SH2 domain-containing signaling proteins represents the activation of different signaling pathways. Therefore, the activation of signaling pathways can be assessed through examination of SH2 domain binding. Comparison of SH2 domain binding of two samples could facilitate the discovery of difference in signaling pathways.

A. Benefits:

• Multiplex - A single assay measures the interaction of RTK with 46 SH2 domains.

• Quantitative - The difference of individual proteins in the interaction of the SH2 domains can be quantitatively compared.

• Simple - The assay is performed the same as the procedure of ELISA.

B. Principle of the technology

SH2 domain-based RTK profiling kit is an ELISA-like assay. SH2 domains are utilized for immobilization on the microtiter wells and an anti-phosphotyrosine antibody along with streptavidin conjugated to horseradish peroxidase (HRP) for detection. Total 46 different SH2 domains are coated in different wells and 46 bindings can be measured. With a 96 well microplate, two samples can be compared among the bindings. The test sample is allowed to react simultaneously with the domain and the antibody. After incubation, the wells are washed to remove unbound-labeled antibody. A HRP substrate, TMB, is added to result in the development of a blue color. The color development is then stopped with the addition of Stop Solution changing the color to yellow. Absorbance is measured spectrophotometrically at 450 nm.

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