Figure 1: Constitutive phosphorylation of Ret in TT cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-Ret (panTyr) Sandwich ELISA Kit #7034 (upper, right). In contrast, a low level of phospho-Ret protein is detected in TT cells lysed in the absence of phosphatase inhibitors* (nonphospho lysate). Similar levels of total Ret protein from both nonphospho or phospho lysates are detected by PathScan® Total Ret Sandwich ELISA Kit #7032 (upper, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using a Phospho-Ret (Tyr905) Rabbit Antibody #3221 (right) or a total Ret (C31B4) Rabbit mAb #3223 (left) are shown in the bottom figure. *Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.
Figure 2: The relationship between protein concentration of phospho or nonphospho lysates and the absorbance at 450 nm is shown. TT cells were cultured (85% confluence) and lysed with or without the addition of phosphatase inhibitor to the lysis buffer (phospho or nonphospho lysate).
|Product Includes||Volume||Solution Color|
|Ret Rabbit mAb Coated Microwells||96 tests|
|Phospho Tyrosine Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-Ret (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated Ret protein. A Ret rabbit mAb has been coated on the microwells. After incubation with cell lysates, Ret protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-tyrosine mouse mAb is added to detect captured tyrosine-phosphorylated Ret protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Ret protein phosphorylated on tyrosine residues.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Phospho-Ret (panTyr) Sandwich ELISA Kit #7034 detects endogenous levels of tyrosine-phosphorylated Ret protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
The Ret proto-oncogene (c-Ret) is a receptor tyrosine kinase that functions as a multicomponent receptor complex in conjunction with other membrane-bound, ligand-binding GDNF family receptors (1). Ligands that bind the Ret receptor include the glial cell line-derived neurotrophic factor (GDNF) and its congeners neurturin, persephin, and artemin (2-4). Research studies have shown that alterations in the corresponding RET gene are associated with diseases including papillary thyroid carcinoma, multiple endocrine neoplasia (type 2A and 2B), familial medullary thyroid carcinoma, and a congenital developmental disorder known as Hirschsprung’s disease (1,3). The Tyr905 residue located in the Ret kinase domain plays a crucial role in Ret catalytic and biological activity. Substitution of Phe for Tyr at position 905 dramatically inhibits Ret autophosphorylation activity (5).
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