Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit #7296

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Kit Includes Volume Solution Color
P-PDGFR alpha (Y849) Ab Coated Microwells 96 tests
PDGFR alpha DetectionAb 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
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

CST's PathScan® Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of PDGF receptor α when phosphorylated at Tyr849. A Phospho-PDGF Receptor α (Tyr849) Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, Phospho-PDGF Receptor α is captured by the coated antibody. Following extensive washing, a PDGFR α Detection Antibody is added to detect the captured phospho-PDGF receptor α 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 PDGF Receptor α phosphorylated on Tyr849.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit #7296 detects PDGF receptor α when phosphorylated at Tyr849. As shown in Figure 1, a significant induction of PDGF Receptor α phosphorylation at Tyr849 can be detected in PDGF-treated MG-63 cells using the PathScan® Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit #7296. The level of total PDGF receptor α (phospho and nonphospho) remains unchanged as shown by Western analysis and by PathScan® Total PDGF Receptor α Sandwich ELISA Kit #7318. 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. Treatment of MG-63 cells with PDGF stimulates tyrosine phosphorylation of PDGF Receptor α as detected by PathScan® Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit #7296, but does not affect the level of total PDGF Receptor α protein detected by either PathScan® Total PDGF Receptor α Sandwich ELISA Kit #7318 or Western analysis. The absorbance readings at 450 nm are shown in the top figure while the corresponding Western blot using PDGF Receptor α (D1E1E) Rabbit mAb #3174 (left) or Phospho-PDGF Receptor α (Tyr849)/PDGF Receptor β (Tyr857) (C43E9) Rabbit mAb #3170 (right) is shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2. The relationship between protein concentration of untreated or PDGF-treated MG-63 cell lysates and the absorbance at 450 nm is shown. Cells were serum starved overnight and then treated with 50 ng/ml PDGF for 7 min. at 37oC.

Background

Platelet derived growth factor (PDGF) family proteins exist as several disulphide-bonded, dimeric isoforms (PDGF AA, PDGF AB, PDGF BB, PDGF CC, and PDGF DD) that bind in a specific pattern to two closely related receptor tyrosine kinases, PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα and PDGFRβ share 75% to 85% sequence homology between their two intracellular kinase domains, while the kinase insert and carboxy-terminal tail regions display a lower level (27% to 28%) of homology (1). PDGFRα homodimers bind all PDGF isoforms except those containing PDGF D. PDGFRβ homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGF receptor α/β binds PDGF B, C, and D homodimers, as well as the PDGF AB heterodimer (2). PDGFRα and PDGFRβ can each form heterodimers with EGFR, which is also activated by PDGF (3). Various cells differ in the total number of receptors present and in the receptor subunit composition, which may account for responsive differences among cell types to PDGF binding (4). Ligand binding induces receptor dimerization and autophosphorylation, followed by binding and activation of cytoplasmic SH2 domain-containing signal transduction molecules, such as GRB2, Src, GAP, PI3 kinase, PLCγ, and NCK. A number of different signaling pathways are initiated by activated PDGF receptors and lead to control of cell growth, actin reorganization, migration, and differentiation (5). Tyr751 in the kinase-insert region of PDGFRβ is the docking site for PI3 kinase (6). Phosphorylated pentapeptides derived from Tyr751 of PDGFRβ (pTyr751-Val-Pro-Met-Leu) inhibit the association of the carboxy-terminal SH2 domain of the p85 subunit of PI3 kinase with PDGFRβ (7). Tyr740 is also required for PDGFRβ-mediated PI3 kinase activation (8).

  1. Deuel, T.F. et al. (1988) Biofactors 1, 213-217.
  2. Bergsten, E. et al. (2001) Nat. Cell Biol. 3, 512-516.
  3. Betsholtz, C. et al. (2001) Bioessays 23, 494-507.
  4. Coughlin, S.R. et al. (1988) Prog. Clin. Biol. Res. 266, 39-45.
  5. Ostman, A. and Heldin, C.H. (2001) Adv. Cancer Res. 80, 1-38.
  6. Panayotou, G. et al. (1992) EMBO J. 11, 4261-4272.
  7. Ramalingam, K. et al. (1995) Bioorg. Med. Chem. 3, 1263-1272.
  8. Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.

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