Figure 1: Treatment of A-204 cells with Human Basic Fibroblast Growth Factor (hFGF basic/FGF2) #8910 stimulates tyrosine phosphorylation of FGFR1, as detected by the PathScan® Phospho-FGF Receptor 1 (panTyr) Sandwich ELISA Kit #12909, but does not affect the level of total FGFR1 detected by PathScan® Total FGF Receptor 1 Sandwich ELISA Kit. Absorbance at 450 nm is shown in the top figure while corresponding western blots using FGF Receptor 1 (D8E4) XP® Rabbit mAb #9740 (left) and Phospho-FGF Receptor (Tyr653/654) Antibody #3471 (right) are shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and hFGF basic-treated A-204 cells and the absorbance at 450 nm as detected by PathScan® Total FGF Receptor 1 Sandwich ELISA Kit is shown. Unstarved A-204 cells (85% confluence) were treated with Human Basic Fibroblast Growth Factor (hFGF basic/FGF2) #8910 (100 ng/ml) for 2-5 min at 37°C and then lysed.
|Product Includes||Volume||Solution Color|
|FGFR1 Rabbit mAb Coated Microwells||96 tests|
|FGF Receptor 1 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® Total FGF Receptor 1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of FGFR1 protein. A FGFR1 rabbit mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-FGFR1 proteins are captured by the coated antibody. Following extensive washing, a FGFR1 mouse antibody is added to detect captured FGFR1 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 absorbance for the developed color is proportional to the quantity of FGFR1 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 FGF Receptor 1 Sandwich ELISA Kit detects endogenous levels of FGFR1 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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