Figure 1. Constitutive phosphorylation of Mer in HCC827 cells lysed in the presence of phosphatase inhibitors (phospho lysate) is detected by PathScan® Phospho-Mer (panTyr) Sandwich ELISA Kit #13340. In contrast, a low level of phospho-Mer protein is detected in HCC827 cells lysed in the absence of phosphatase inhibitors (nonphospho lysate). Similar levels of total Mer protein from both nonphospho and phospho lysates are detected by PathScan® Total Mer Sandwich ELISA Kit #13488 (upper, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using Mer (D21F11) XP® Rabbit mAb #4319 (left) and a phospho-Mer (Tyr749/753) antibody (right) are shown in the bottom figure. Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate.
Figure 2. The relationship between protein concentration of phospho or nonphospho lysates and the absorbance at 450 nm as detected by the PathScan® Total Mer Sandwich ELISA Kit is shown. Unstarved HCC827 cells were cultured (85% confluence) and lysed with or without addition of phosphatase inhibitors to the lysis buffer (phospho or nonphospho lysate, respectively).
PathScan® Total Mer Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Mer protein. A Mer Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, Mer protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Mer Mouse Detection mAb is added to detect the captured Mer proteins. 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 optical density for this developed color is proportional to the quantity of Mer protein.
Antibodies in kit are custom formulations specific to kit.
PathScan® Total Mer Sandwich ELISA Kit recognizes endogenous levels of Mer 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.
Mer tyrosine kinase belongs to a receptor tyrosine kinase family with Axl and Tyro3. This family is characterized by a common NCAM (neural adhesion molecule)-related extracellular domain and a common ligand, GAS6 (growth arrest-specific protein 6). Mer protein has an apparent molecular weight of 170-210 kDa due to different glycosylation patterns generated in different cell types. Mer can be activated by dimerization and autophosphorylation through ligand binding or homophilic cell-cell interaction mediated by its NCAM-like motif (1). The downstream signaling components of activated Mer include PI3 kinase, PLCγ, and MAP kinase (2). Family members are prone to transcriptional regulation and carry out diverse functions including the regulation of cell adhesion, migration, phagocytosis, and survival (3). Mer regulates macrophage activation, promotes apoptotic cell engulfment, and supports platelet aggregation and clot stability in vivo (4). Investigators have found that overexpression of Mer may play a cooperative role in leukemogenesis and may be an effective target for biologically based leukemia/lymphoma therapy (5).
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