Figure 1: Constitutive phosphorylation of ROS1 in HCC78 cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-ROS1 (panTyr) Sandwich ELISA Kit #7093 (upper, right). In contrast, a low level of phospho-ROS1 protein is detected in HCC78 cells lysed in the absence of phosphatase inhibitors* (nonphospho lysate). Similar levels of total HCC78 protein from both nonphospho and phospho lysates are detected by PathScan® Total ROS1 Sandwich ELISA Kit #7091 (upper, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using Phospho-ROS1 (Tyr2274) Antibody #3078 (right) or total ROS1 (69D6) Mouse mAb #3266 (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. 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).
|Product Includes||Volume||Solution Color|
|ROS1 Mouse mAb Coated Microwells||96 tests|
|ROS1 Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit 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|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total ROS1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of ROS1 protein. A ROS1 mouse antibody has been coated onto the microwells. After incubation with cell lysates, ROS1 protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a ROS1 rabbit detection antibody is added to detect captured ROS1 protein. Anti-rabbit 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.
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® Total ROS1 Sandwich ELISA Kit #7091 detects endogenous levels of ROS1 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.
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).
Explore pathways + proteins related to this product.