Product Pathways - PathScan ELISA

PathScan^® Total Ret Sandwich ELISA Kit #7032

Kit Includes	Volume	Solution Color
Ret Rabbit mAb Coated Microwells	96 tests
Ret Mouse Detection mAb	11 milliliters	Green
Anti-mouse IgG, HRP-linked Antibody	11 milliliters	Red
TMB Substrate	11 milliliters	Colorless
STOP Solution	11 milliliters	Colorless
20X Wash Buffer	25 milliliters	Colorless
Sample Diluent	25 milliliters	Blue
Cell Lysis Buffer (10X) # 9803	15 milliliters	Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

Reactivity Key: H=Human

Description

The PathScan^® Total Ret Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total 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 Ret mouse mAb is added to detect captured 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 total Ret protein.

Specificity / Sensitivity

PathScan^® Total Ret Sandwich ELISA Kit #7032 detects endogenous levels of total Ret protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2.

ELISA - Western correlation

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.

Sensitivity

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).

Background

The Ret proto-oncogene (c-Ret) is a receptor tyrosine kinase that functions as a multicompetent 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 neurotropic factor (GDNF) and its congeners neurturin, persephin and artemin (2-4). 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 Tyr905 dramatically inhibits Ret autophosphorylation activity (5).

Application References

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Companion Products

Rabbit monoclonal antibody is produced under license (granting certain rights including those under U. S. Patent No. 5,675,063) from Epitomics, Inc.

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.