Figure 1: Treatment of Hep G2 cells with Human FGF acidic (hFGF acidic) #5234 stimulates tyrosine phosphorylation of FGF receptor 4 protein, which can be detected by the PathScan® Phospho-FGF Receptor 4 (panTyr) Sandwich ELISA Kit. This kit can also detect the high level of tyrosine-phosphorylated FGF receptor 4 from CHO cells that are transfected with human FGF receptor 4 protein. The absorbance readings at 450 nm are shown in the top figure while corresponding western blots using FGF Receptor 4 (D3B12) XP® Rabbit mAb #8562 (left) and Phospho-FGF Receptor (Tyr653/654) (55H2) Mouse mAb #3476 (right) are shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and hFGF-treated Hep G2 cells and the absorbance at 450 nm as detected by the PathScan® Phospho-FGF Receptor 4 (panTyr) Sandwich ELISA Kit #69193 is shown. Starved Hep G2 cells (85% confluence) were treated with Human FGF acidic (hFGF acidic) #5234 (100 ng/ml, 2-5 min, 37°C) and then lysed.
|Product Includes||Volume||Solution Color|
|FGFR4 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|
PathScan® Phospho-FGF Receptor 4 (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated FGR receptor 4 protein. An FGF receptor 4 rabbit mAb has been coated onto the microwells. After incubation with cell lysates, FGF receptor 4 protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-tyrosine mouse detection mAb is added to detect captured phospho-FGF receptor 4 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 optical density for this developed color is proportional to the quantity of FGF Receptor 4 protein phosphorylated on tyrosine residues.
Antibodies in the 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-FGF Receptor 4 (panTyr) Sandwich ELISA Kit recognizes endogenous levels of tyrosine-phosphorylated FGF receptor 4 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.
Fibroblast growth factors (FGFs) produce mitogenic and angiogenic effects in target cells by signaling through cell surface receptor tyrosine kinases. There are four members of the FGF receptor family: FGFR1 (flg), FGFR2 (bek, KGFR), FGFR3, and FGFR4. Each receptor contains an extracellular ligand binding domain, a transmembrane domain, and a cytoplasmic kinase domain (1). Following ligand binding and dimerization, the receptors are phosphorylated at specific tyrosine residues (2). Seven tyrosine residues in the cytoplasmic tail of FGFR1 can be phosphorylated: Tyr463, 583, 585, 653, 654, 730, and 766. Tyr653 and Tyr654 are important for catalytic activity of activated FGFR and are essential for signaling (3). The other phosphorylated tyrosine residues may provide docking sites for downstream signaling components such as Crk and PLCγ (4,5).
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