Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-ROS1 (panTyr) Sandwich ELISA Kit #7093

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Important Ordering Details: Product is assembled upon order to ensure maximum activity. Domestic: Please allow up to two weeks for your order to be processed and shipped. International: Please allow up to three weeks, depending on the country, for your order to be processed and shipped.

Kit Includes Volume Solution Color
ROS1 Rabbit mAb coated microwells 96 tests
Phospho-Tyrosine Mouse Detection Antibody 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml 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, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST's PathScan® Phospho-Ros (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated Ros protein. A Ros rabbit antibody has been coated on the microwells. After incubation with cell lysates, Ros protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-tyrosine mouse detection antibody is added to detect captured tyrosine-phosphorylated Ros 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 Ros protein phosphorylated on tyrosine.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-Ros (panTyr) Sandwich ELISA Kit #7093 detects endogenous levels of Ros protein phosphorylated at Tyr residues 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.

ELISA - Western correlation

ELISA - Western correlation

Figure 1: Constitutive phosphorylation of Ros in HCC78 cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-Ros (panTyr) Sandwich ELISA Kit #7093 (upper, right). In contrast, a low level of phospho-Ros protein is detected in HCC78 cells lysed in the absence of phosphatase inhibitors* (nonphospho lysate). Similar levels of total Ros protein from both nonphospho and phospho lysates are detected by PathScan® Total Ros Sandwich ELISA Kit #7091 (upper, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using a Phospho-Ros (Tyr2274) Antibody #3078 (right) or a total Ros Mouse mAb #3266 (left) are shown in the bottom figure. *Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of phospho or nonphospho lysates and the absorbance at 450 nm is shown. HCC78 cells were cultured (85% confluence) and lysed with or without the addition of phosphatase inhibitor to the lysis buffer (phospho or nonphospho lysate, respectively).

Background

ROS1, an orphan receptor tyrosine kinase of the insulin receptor family, was initially identified as a homolog of v-ros from the UR2 sarcoma virus (1). ROS1 consists of a large extracellular domain that is composed of six fibronectin repeats, a transmembrane domain, and an intracellular kinase domain. While the function of ROS1 is undefined, it has been shown to play an important role in differentiation of epididymal epithelium (2). The first oncogenic fusion of ROS1, FIG-ROS1, was initially identified by research studies in glioblastoma (3), and subsequent studies have found this fusion in cholangiocarcinoma (4), ovarian cancer (5) and non-small cell lung cancer (NSCLC) (6). Investigators have found additional oncogenic ROS1 fusion proteins in NSCLC (at a frequency of ~1.6%), where the ROS1 kinase domain is fused to the amino-terminal region of a number of different proteins, including CD74 and SLC34A2 (6-8). ROS1 fusion proteins activate the SHP-2 phosphatase, PI3K/Akt/mTOR, Erk, and Stat3 pathways (3,4,9).

  1. Matsushime, H. et al. (1986) Mol Cell Biol 6, 3000-4.
  2. Yeung, C.H. et al. (1999) Biol Reprod 61, 1062-9.
  3. Charest, A. et al. (2003) Genes Chromosomes Cancer 37, 58-71.
  4. Gu, T.L. et al. (2011) PLoS One 6, e15640.
  5. Birch, A.H. et al. (2011) PLoS One 6, e28250.
  6. Rimkunas, V.M. et al. (2012) Clin Cancer Res 18, 4449-57.
  7. Rikova, K. et al. (2007) Cell 131, 1190-203.
  8. Stumpfova, M. and Jänne, P.A. (2012) Clin Cancer Res 18, 4222-4.
  9. Jun, H.J. et al. (2012) Cancer Res 72, 3764-74.

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