Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-FGFR2 (panTyr) Sandwich ELISA Kit #7954

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Kit Includes Volume Solution Color
FGFR2 Rabbit Antibody 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

The PathScan® Phospho-FGFR2 (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated FGFR2 protein. An FGFR2 rabbit antibody has been coated on the microwells. After incubation with cell lysates, FGFR2 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 FGFR2 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 tyrosine-phosphorylated FGFR2 protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-FGFR2 (panTyr) Sandwich ELISA Kit #7954 detects endogenous levels of FGFR2 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 FGFR2 in KATO III cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-FGFR2 (panTyr) Sandwich ELISA Kit #7954 (upper, right). In contrast, a low level of phospho-FGFR2 protein is detected in KATO III cells lysed in the absence of phosphatase inhibitors (nonphospho lysate). Similar levels of total FGFR2 protein from both nonphospho and phospho lysates are detected by PathScan® Total FGFR2 Sandwich ELISA Kit #7939 (upper, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using Phospho-FGF Receptor (Tyr653/654) Antibody #3471 (right) or a total FGFR2 Rabbit mAb (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. KATO III 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

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).

  1. Powers, C.J. et al. (2000) Endocr Relat Cancer 7, 165-97.
  2. Reilly, J.F. et al. (2000) J Biol Chem 275, 7771-8.
  3. Mohammadi, M. et al. (1996) Mol Cell Biol 16, 977-89.
  4. Mohammadi, M. et al. (1991) Mol Cell Biol 11, 5068-78.
  5. Larsson, H. et al. (1999) J Biol Chem 274, 25726-34.

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